Stabilized bioactive peptides and methods of identification, synthesis, and use

ABSTRACT

An intracellular selection system allows screening for peptide bioactivity and stability. Randomized recombinant peptides are screened for bioactivity in a tightly regulated expression system, preferably derived from the wild-type lac operon. Bioactive peptides thus identified are inherently protease- and peptidase-resistant. Also provided are bioactive peptides stabilized by a stabilizing group at the N-terminus, the C-terminus, or both. The stabilizing group can be a small stable protein, such as the Rop protein, glutathione sulfotransferase, thioredoxin, maltose binding protein, or glutathione reductase, an α-helical moiety, or one or more proline residues.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.09/701,947, filed Dec. 5, 2000, which is a National Stage applicationunder 35 U.S.C. §371 of PCT/US99/23731, filed Oct. 12, 1999, which inturn claims the benefit of U.S. Provisional Patent Applications SerialNos. 60/104,013, filed Oct. 13, 1998, and 60/112,150, filed Dec. 14,1998, each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This invention relates to stabilized bioactive peptides, and moreparticularly to bioactive peptides that contain heterologous stabilizinggroups attached to one or both of the bioactive peptide's termini.

BACKGROUND

[0003] Bioactive peptides are small peptides that elicit a biologicalactivity. Since the discovery of secretin in 1902, over 500 of thesepeptides which average 20 amino acids in size have been identified andcharacterized. They have been isolated from a variety of systems,exhibit a wide range of actions, and have been utilized as therapeuticagents in the field of medicine and as diagnostic tools in both basicand applied research. Tables 1 and 2 list some of the best knownbioactive peptides. TABLE 1 Bioactive peptides utilized in medicine Sizein Amino Name Isolated From Acids Therapeutic Use Angiotensin II HumanPlasma 8 Vasoconstrictor Bradykinin Human Plasma 9 Vasodilator CaeruleinFrom Skin 10 Choleretic Agent Calcitonin Human Parathyroid Gland 32Calcium Regulator Cholecystokinin Porcine Intestine 33 Choleretic AgentCorticotropin Porcine Pituitary Gland 39 Hormone Eledoisin Octopod Venom11 Hypotensive Agent Gastrin Porcine Stomach 17 Gastric ActivatorGlucagon Porcine Pancreas 29 Antidiabetic Agent Gramicidin D Bacillusbrevis Bacteria 11 Antibacterial Agent Insulin Canine PancreasAntidiabetic Agent Insulin A 21 Insulin B 30 Kallidin Human Plasma 10Vasodilator Luteinizing Bovine Hypothalamus 10 Hormone Hormone-ReleasingStimulator Factor Melittin Bee Venom 26 Antirheumatic Agent OxytocinBovine Pituitary Gland 9 Oxytocic Agent Secretin Canine Intestine 27Hormone Sermorelin Human Pancreas 29 Hormone Stimulator SomatostatinBovine Hypothalamus 14 Hormone Inhibitor Vasopressin Bovine PituitaryGland 9 Antidiuretic Agent

[0004] TABLE 2 Bioactive peptides utilized in applied research Size inAmino Name Isolated From Acids Biological Activity Atrial NatriureticRat Atria 28 Natriuretic Agent Peptide Bombesin Frog Skin 14 GastricActivator Conantokin G Snail Venom 17 Neurotransmitter Conotoxin G1Snail Venom 13 Neuromuscular Inhibitor Defensin HNP-1 Human Neutrophils30 Antimicrobial Agent Delta Sleep- Rabbit Brain 9 Neurological AffectorInducing Peptide Dermaseptin Frog Skin 34 Antimicrobial Agent DynorphinPorcine Brain 17 Neurotransmitter EETI II Ecballium elaterium 29Protease Inhibitor seeds Endorphin Human Brain 30 NeurotransmitterEnkephalin Human Brain 5 Neurotransmitter Histatin 5 Human Saliva 24Antibacterial Agent Mastoparan Vespid Wasps 14 Mast Cell DegranulatorMagainin 1 Frog Skin 23 Antimicrobial Agent Melanocyte Porcine Pituitary13 Hormone Stimulator Stimulating Gland Hormone Motilin Canine Intestine22 Gastric Activator Neurotensin Bovine Brain 13 NeurotransmitterPhysalaemin Frog Skin 11 Hypotensive Agent Substance P Horse Intestine11 Vasodilator Vasoactive Porcine Intestine 28 Hormone IntestinalPeptide

[0005] Where the mode of action of these peptides has been determined,it has been found to be due to the interaction of the bioactive peptidewith a specific protein target. In most of the cases, the bioactivepeptide acts by binding to and inactivating its protein target withextremely high specificities. Binding constants of these peptides fortheir protein targets typically have been determined to be in thenanomolar (nM, 10⁻⁹ M) range with binding constants as high as 10⁻¹² M(picomolar range) having been reported. Table 3 shows target proteinsinactivated by several different bioactive peptides as well as thebinding constants associated with binding thereto. TABLE 3 Bindingconstants of bioactive peptides Size in Amino Binding Bioactive PeptideAcids Inhibited Protein Constant α-Conotoxin GIA 15 Nicotinic 1.0 ×10⁻⁹M Acetylcholine EETI II 29 Trypsin 1.0 × 10⁻¹²M H2 (7-5)  8 HSVRibonucleotide 3.6 × 10⁻⁵M Reductase Histatin 5 24 Bacteroidesgingivalis 5.5 × 10⁻⁸M Protease Melittin 26 Calmodulin 3.0 × 10⁻⁹MMyotoxin (29-42) 14 ATPase 1.9 × 10⁻⁵M Neurotensin 13 Ni RegulatoryProtein 5.6 × 10⁻¹¹M Pituitary Adenylate 38 Calmodulin 1.5 × 10⁻⁸MCyclase Activating Polypeptide PKI (5-24) 20 CAMP-Dependent 2.3 × 10⁻⁹MProtein Kinase SCP (153-180) 27 Calpain 3.0 × 10⁻⁸M Secretin 27 HSR GProtein 3.2 × 10⁻⁹M Vasoactive Intestinal 28 GPRNI G Protein 2.5 × 10⁻⁹MPeptide

[0006] Recently, there has been an increasing interest in employingsynthetically derived bioactive peptides as novel pharmaceutical agentsdue to the impressive ability of the naturally occurring peptides tobind to and inhibit specific protein targets. Synthetically derivedpeptides could be useful in the development of new antibacterial,antiviral, and anticancer agents. Examples of synthetically derivedantibacterial or antiviral peptide agents would be those capable ofbinding to and preventing bacterial or viral surface proteins frominteracting with their host cell receptors, or preventing the action ofspecific toxin or protease proteins. Examples of anticancer agents wouldinclude synthetically derived peptides that could bind to and preventthe action of specific oncogenic proteins.

[0007] To date, novel bioactive peptides have been engineered throughthe use of two different in vitro approaches. The first approachproduces candidate peptides by chemically synthesizing a randomizedlibrary of 6-10 amino acid peptides (J. Eichler et al., Med. Res. Rev.15: 48 1-496 (1995); K. Lam, Anticancer Drug Des. 12:145-167 (1996); M.Lebl et al., Methods Enzymol. 289:336-392 (1997)). In the secondapproach, candidate peptides are synthesized by cloning a randomizedoligonucleotide library into a Ff filamentous phage gene, which allowspeptides that are much larger in size to be expressed on the surface ofthe bacteriophage (H. Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424 (1997); G. Smith et al., et al. Meth. Enz. 217: 228-257 (1993)).To date, randomized peptide libraries up to 38 amino acids in lengthhave been made, and longer peptides are likely achievable using thissystem. The peptide libraries that are produced using either of thesestrategies are then typically mixed with a preselected matrix-boundprotein target. Peptides that bind are eluted, and their sequences aredetermined. From this information new peptides are synthesized and theirinhibitory properties are determined. This is a tedious process thatonly screens for one biological activity at a time.

[0008] Although these in vitro approaches show promise, the use ofsynthetically derived peptides has not yet become a mainstay in thepharmaceutical industry. The primary obstacle remaining is that ofpeptide instability within the biological system of interest asevidenced by the unwanted degradation of potential peptide drugs byproteases and/or peptidases in the host cells. There are three majorclasses of peptidases which can degrade larger peptides: amino andcarboxy exopeptidases which act at either the amino or the carboxyterminal end of the peptide, respectively, and endopeptidases which acton an internal portion of the peptide. Aminopeptidases,carboxypeptidases, and endopeptidases have been identified in bothprokaryotic and eukaryotic cells. Many of those that have beenextensively characterized were found to function similarly in both celltypes. Interestingly, in both prokaryotic and eukaryotic systems, manymore arninopeptidases than carboxypeptidases have been identified todate.

[0009] Approaches used to address the problem of peptide degradationhave included the use of D-amino acids or modified amino acids asopposed to the naturally occurring L-amino acids (e.g., J. Eichler etal., Med Res Rev. 15:48 1496 (1995); L. Sanders, Eur. J. Drug Metabol.Pharmacokinetics 15:95-102 (1990)), the use of cyclized peptides (e.g.,R. Egleton, et al., Peptides 18:1431-1439 (1997)), and the developmentof enhanced delivery systems that prevent degradation of a peptidebefore it reaches its target in a patient (e. g., L. Wearley, Crit. Rev.Ther. Drug Carrier Syst. 8:33 1-394 (1991); L. Sanders, Eur. J. DrugMetabol. Pharmacokinetics 15: 95-102 (1990)). Although these approachesfor stabilizing peptides and thereby preventing their unwanteddegradation in the biosystem of choice (e.g., a patient) are promising,there remains no way to routinely and reliably stabilize peptide drugsand drug candidates. Moreover, many of the existing stabilization anddelivery methods cannot be directly utilized in the screening anddevelopment of novel useful bioactive peptides. A biological approachthat would serve as both a method of stabilizing peptides and a methodfor identifying novel bioactive peptides would represent a much neededadvance in the field of peptide drug development.

SUMMARY OF THE INVENTION

[0010] The present invention provides an intracellular screening methodfor identifying novel bioactive peptides. A host cell is transformedwith an expression vector comprising a tightly regulable control regionoperably linked to a nucleic acid sequence encoding a peptide.Typically, the encoded peptide has a stabilizing group positioned at oneor both ends of the peptide. The transformed host cell is first grownunder conditions that repress expression of the peptide and then,subsequently, expression of the peptide is induced. Phenotypic changesin the host cell upon expression of the peptide are indicative ofbioactivity, and are evaluated. If, for example, expression of thepeptide is accompanied by complete or partial inhibition of host cellgrowth, the expressed peptide constitutes a bioactive peptide, in thatit functions as an inhibitory peptide.

[0011] Intracellular identification of bioactive peptides can beadvantageously carried out in a pathogenic microbial host cell.Bioactive peptides having antimicrobial activity are readily identifiedin a microbial host cell system. Further, the method can be carried outin a host cell that has not been modified to reduce or eliminate theexpression of naturally expressed proteases or peptidases. When carriedout in a host cell comprising proteases and peptides, the selectionprocess of the invention is biased in favor of bioactive peptides thatare protease and peptidase-resistant.

[0012] The tightly regulable control region of the expression vectorused to transform the microbial host cell according to the invention canbe derived from the wild-type Escherichia coli lac operon, and thetransformed host cell can include an amount of Lac repressor proteineffective to repress expression of the peptide during host cell growthunder repressed conditions. To insure a sufficient amount of Lacrepressor protein, the host cell can be transformed with a second vectorthat overproduces Lac repressor protein.

[0013] Optionally, the expression vector used to transform the host cellcan be genetically engineered to encode a stabilized peptide that isresistant to peptidases and proteases. For example, the coding sequencecan be designed to encode a stabilizing group at either or both of thepeptide's N-terminus or C-terminus. As another example, the codingsequence can be designed to encode a stabilizing motif such as anα-helix motif or an opposite charge ending motif, as described below.The presence of a stabilizing group at a peptide terminus and/or of astabilizing motif can slow down the rate of intracellular degradation ofthe peptide.

[0014] A plurality of vectors can be used to screen a randomized libraryof candidate bioactive peptides.

[0015] The present invention also provides a polypeptide that includes abioactive peptide and a stabilizing group coupled to at least oneterminus of the bioactive peptide. Preferably, the bioactive peptide is50 or fewer amino acids in length. The stabilizing group is heterologousto the bioactive peptide and can be, for example, a proline, aproline-containing peptide, a single α-helix or multiple helix bundle,or other polypeptide or small protein such as Rop, human serum albumin,and the like. In one embodiment of the stabilized polypeptide, thestabilizing group(s) lack the capacity to participate in the formationof an intramolecular disulfide bond within the polypeptide. Thus, thestabilizing group is preferably not a thioredoxin polypeptide.

[0016] When a polypeptide includes a stabilizing group on each terminus,the stabilizing groups can be the same or different. If different, thestabilizing groups are optionally heterologous to each other, as thatterm is defined below. The first and second stabilizing groups can, butneed not, interact to form a naturally occurring secondary or tertiarystructure. Further, the first and second stabilizing groups can, butneed not, confine the N-terminus and the C-terminus of the bioactivepeptide in close proximity.

[0017] The invention further includes a nucleic acid encoding thepolypeptide of the invention. A vector that contains such a nucleic acidis also included. Preferably the vector contains a tightly regulableexpression control sequence operably linked to the nucleic acid sequenceencoding the stabilized polypeptide.

[0018] The present invention also includes a method for making astabilized polypeptide that involves coupling a stabilizing group to atleast one terminus of a bioactive peptide. Coupling can be achievedchemically or enzymatically, or can occur as the result of translationin a host cell of a vector containing a nucleic acid sequence encodingthe stabilized polypeptide. The vector comprises an expression controlsequence operably linked to the coding sequence; preferably, theexpression control sequence is tightly regulable in said host cell.Optionally the method includes determining stability of said stabilizedpolypeptide relative to said bioactive peptide.

[0019] When the method is performed in a host cell, the host cell isfirst transformed with an exogenous nucleic acid encoding the stabilizedpolypeptide, then the stabilized polypeptide is expressed and recovered.The host cells can be prokaryotic, such as bacteria, or eukaryotic.

[0020] Phage display can be used to identify a bioactive peptide thatcan be subsequently stabilized according to the invention. Whendisplayed on the surface of a bacteriophage, bioactive peptides aretethered at one end by a bacteriophage protein. The free bioactivepeptide (i.e., uncoupled from the bacteriophage protein) may exhibit alack of stability in vivo. Hence, the invention involves stabilizingthese bioactive peptides by coupling them to a stabilizing group at theend that had been tethered during phage display, thereby effectivelyreplacing the bacteriophage protein as a stabilizing feature. Couplingcan take place chemically, enzymatically, or by way of recombinantgenetic engineering, as described herein. Polypeptides thus stabilizedare also included in the invention.

[0021] Alternatively, the stabilized polypeptide can be produced as adirect product of phage display. A bacteriophage that contains anexogenous nucleic acid encoding a polypeptide comprising a bioactivepeptide (or candidate bioactive peptide), a bacteriophage proteincoupled to one terminus of the bioactive peptide, and a stabilizinggroup coupled to the other terminus of the bioactive peptide is culturedunder conditions to cause the bacteriophage to express the stabilizedpolypeptide and display it on its surface. The stabilizing group can becoupled to either end of the bioactive peptide, and the bacteriophageprotein is coupled to the other end. Optionally the stabilizedpolypeptide is cleaved from the host cell surface to yield a stabilizedbioactive peptide comprising the bioactive peptide and the stabilizinggroup.

[0022] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0023] Other features and advantages of the invention will be apparentfrom the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 shows the control region (SEQ ID NO: 1) of the wild-typelac operon from the auxiliary operator O3 through the translationalstart of the lacZ gene. DNA binding sites include the operators O3 andO1 (both underlined), CAP (boxed), the −35 site (boxed), and the −10site (boxed), while important RNA and protein sites include the Lacltranslation stop site (TGA), the +1 lacZ transcription start site, theShine Dalgarno (SD) ribosome binding site for lacZ, and the LacZtranslation start site (ATG).

[0025]FIG. 2 is a map of plasmid pLAC11. The unique restriction sitesand the base pair at which they cut are indicated. Other sites ofinterest are also shown, including Tet (98-1288), Rop (1931-2122), ori(2551-3138), Amp (3309-25 4 169), and lacPO (4424-4536).

[0026]FIG. 3 is a map of plasmid pLAC22. The unique restriction sitesand the base pair at which they cut are indicated. Other sites ofinterest are also shown, including Tet (98-1288), Rop (1927-2118), ori(2547-3134), Amp (3305-4165), lacIq (4452-5536), and lacPO (5529-5641).

[0027]FIG. 4 is a map of plasmid pLAC33. The unique restriction sitesand the base pair at which they cut are indicated. Other sites ofinterest are also shown, including Tet (98-1288), ori (1746-2333), Amp(2504-3364), and lacPO (3619-3731).

[0028]FIG. 5 shows the response of the pLAC11-lacZ construct (opencircles) to varying amounts of isopropyl β-D-thiogalactoside (IPTG). Afilled square indicates the β-galactosidase activity that was obtainedwhen MG1655 or CSH27 cells were grown in rich media induced with 1 mMIPTG, while a filled diamond indicates the β-galactosidase activity thatwas obtained when MG1655 or CSH27 cells were grown in M9 minimal lactosemedia.

[0029]FIG. 6 shows growth curves depicting the inhibitory effects of atwo day inhibitor (pPep12) versus a one day inhibitor (pPep1). Datapoints for the control, pLAC 11, for pPep 1, and for pPep 12, areindicated by squares, circles, and triangles, respectively.

[0030]FIG. 7 is a map of the p-Rop(C) fusion vector. The uniquerestriction sites and the base pair at which they cut are indicated.Other sites of interest are also shown, including Rop (7-198), ori(627-1214), Amp (2245-1385), lacPO (2500-26 12).

[0031]FIG. 8 is a map of the p(N)Rop-fusion vector. The uniquerestriction sites and the base pair at which they cut are indicated.Other sites of interest are also shown: Rop (7-204), ori (266-853), Amp(1024-1 884), lacPO (2139-2251).

[0032]FIG. 9 illustrates a peptide (SEQ ID NO:2) having the oppositecharge ending motif, wherein the amino and carboxy termini of thepeptide are stabilized by the interactions of the opposite charge endingamino acids.

DETAILED DESCRIPTION

[0033] The present invention represents a significant advance in the artof peptide drug development by allowing concurrent screening for peptidebloactivity and stability. Randomized recombinant peptides are screenedfor bioactivity in a tightly regulated inducible expression system thatpermits essentially complete repression of peptide expression in thehost cell. Subsequent induction of peptide expression can then be usedto identify peptides that inhibit host cell growth or possess otherbioactivities.

[0034] Intracellular screening of randomized peptides has manyadvantages over existing methods. Bioactivity is readily apparent, manydiverse bioactivities can be screened for simultaneously, very largenumbers of peptides can be screened using easily generated peptidelibraries, and the host cell, if desired, can be genetically manipulatedto identify an affected protein target. Advantageously, randomizedpeptides can be screened in a host cell that is identical to or closelyresembles the eventual target cell for antimicrobial, anticancer, andother therapeutic applications. An additional and very important featureof this system is that selection is naturally biased in favor ofpeptides that are stable in an intracellular environment, i.e., that areresistant to proteases and peptidases. Fortuitously, bacterialpeptidases are very similar to eukaryotic peptidases. Peptides that arestable in a bacterial host are thus likely to be stable in a eukaryoticcell as well, allowing bacterial cells to be used in initial screens toidentify drugs that may eventually prove useful as human or animaltherapeutics.

[0035] The invention is directed to the identification and use ofbioactive peptides. A bioactive peptide is a peptide having a biologicalactivity. The term “bioactivity” as used herein includes, but is notlimited to, any type of interaction with another biomolecule, such as aprotein, glycoprotein, carbohydrate, for example an oligosaccharide orpolysaccharide, nucleotide, polynucleotide, fatty acid, hormone, enzyme,cofactor or the like, whether the interactions involve covalent ornoncovalent binding. Bioactivity further includes interactions of anytype with other cellular components or constituents including salts,ions, metals, nutrients, foreign or exogenous agents present in a cellsuch as viruses, phage and the like, for example binding, sequestrationor transport-related interactions. Bioactivity of a peptide can bedetected, for example, by observing phenotypic effects in a host cell inwhich it is expressed, or by performing an in vitro assay for aparticular bioactivity, such as affinity binding to a target molecule,alteration of an enzymatic activity, or the like. Examples of bioactivepeptides include antimicrobial peptides and peptide drugs. Antimicrobialpeptides are peptides that adversely affect a microbe such as abacterium, virus, protozoan, or the like. Antimicrobial peptidesinclude, for example, inhibitory peptides that slow the growth of amicrobe, microbiocidal peptides that are effective to kill a microbe(e.g., bacteriocidal and virocidal peptide drugs, sterilants, anddisinfectants), and peptides effective to interfere with microbialreproduction, host toxicity, or the like. Peptide drugs for therapeuticuse in humans or other animals include, for example, antimicrobialpeptides that are not prohibitively toxic to the patient, and peptidesdesigned to elicit, speed up, slow down, or prevent various metabolicprocesses in the host such as insulin, oxytocin, calcitonin, gastrin,somatostatin, anticancer peptides, and the like.

[0036] The term “peptide” as used herein refers to a plurality of aminoacids joined together in a linear chain via peptide bonds. Accordingly,the term “peptide” as used herein includes a dipeptide, tripeptide,oligopeptide and polypeptide. A dipeptide contains two amino acids; atripeptide contains three amino acids; and the term oligopeptide istypically used to describe peptides having between 2 and about 50 ormore amino acids. Peptides larger than about 50 are often referred to aspolypeptides or proteins. For purposes of the present invention, a“peptide” is not limited to any particular number of amino acids.Preferably, however, the peptide contains about 2 to about 50 aminoacids (e.g., about 5 to about 40 amino acids, about 5 to about 20 aminoacids, or about 7 to 15 amino acids).

[0037] The library used to transform the host cell is formed by cloninga randomized, peptide-encoding oligonucleotide into a nucleic acidconstruct having a tightly regulable expression control region. Anexpression control region can be readily evaluated to determine whetherit is “tightly regulable,” as the term is used herein, by bioassay in ahost cell engineered to contain a mutant nonfunctional gene “X.”Transforming the engineered host cell with an expression vectorcontaining a tightly regulable expression control region operably linkedto a cloned wild-type gene “X” will preserve the phenotype of theengineered host cell under repressed conditions. Under inducedconditions, however, the expression vector containing the tightlyregulable expression control region that is operably linked to thecloned wild-type gene “X” will complement the mutant nonfunctional geneX to yield the wild-type phenotype. In other words, a host cellcontaining a null mutation which is transformed with a tightly regulableexpression vector capable of expressing the chromosomally inactivatedgene will exhibit the null phenotype under repressed conditions; butwhen expression is induced, the cell will exhibit a phenotypeindistinguishable from the wild-type cell. It should be understood thatthe expression control region in a tightly regulable expression vectorof the present invention can be readily modified to produce higherlevels of an encoded biopeptide, if desired (see, e.g., Example 1,below). Such modification may unavoidably introduce some “leakiness”into expression control, resulting in a low level of peptide expressionunder repressed conditions.

[0038] In one embodiment, the expression control region of the inducibleexpression vector is derived from the wild-type E. coli lacpromoter/operator region. The expression vector can contain a regulatoryregion that includes the auxiliary operator O3, the CAP binding region,the −35 promoter site, the −10 promoter site, the operator O1, theShine-Dalgarno sequence for lacZ, and a spacer region between the end ofthe Shine-Dalgarno sequence and the ATG start of the lacZ codingsequence (see FIG. 1).

[0039] It is to be understood that variations in the wild-type nucleicacid sequence of the lac promoter/operator region can be tolerated inthe expression control region of the preferred expression vector and areencompassed by the invention, provided that the expression controlregion remains tightly regulable as defined herein. For example, the −10site of the wild-type lac operon (TATGTT) is weak compared to thebacterial consensus −10 site sequence TATAAT, sharing four out of sixpositions. It is contemplated that other comparably weak promoters areequally effective at the −10 site in the expression control region; astrong promoter is to be avoided in order to insure complete repressionin the uninduced state. With respect to the −35 region, the sequence ofthe wild-type lac operon, TTTACA, is one base removed from the consensus−35 sequence TTGACA. It is contemplated that a tightly regulable lacoperon-derived expression control region could be constructed using aweaker −35 sequence (i.e., one having less identity with the consensus−35 sequence) and a wild-type −10 sequence (TATAAT), yielding a weakpromoter that needs the assistance of the CAP activator protein.Similarly, it is to be understood that the nucleic acid sequence of theCAP binding region can be altered as long as the CAP protein binds to itwith essentially the same affinity. The spacer region between the end ofthe Shine-Dalgarno sequence and the ATG start of the lacZ codingsequence is typically between about 5 and about 10 nucleotides inlength, preferably about 5 to about 8 nucleotides in length, morepreferably about 7-9 nucleotides in length. The most preferredcomposition and length of the spacer region depends on the compositionand length of Shine-Dalgarno sequence with which it is operably linkedas well as the translation start codon employed (i.e., AUG, GUG, orUUG), and can be determined accordingly by one of skill in the art.Preferably the nucleotide composition of the spacer region is “AT rich”;that is, it contains more A's and T's than it does G's and C's.

[0040] In one embodiment of the method of the invention, the expressionvector has the identifying characteristics of pLAC11 (ATCC No. 207108)and, in a particularly convenient embodiment, is pLAC11 (ATCC No.207108).

[0041] As used in the present invention, the term “vector” is to bebroadly interpreted as including a plasmid, including an episome, aviral vector, a cosmid, or the like. A vector can be circular or linear,single-stranded or double-stranded, and can comprise RNA, DNA, ormodifications and combinations thereof. Selection of a vector or plasmidbackbone depends upon a variety of characteristics desired in theresulting construct, such as selection marker(s), plasmid copy number,and the like. A nucleic acid sequence is “operably linked” to anexpression control sequence in the regulatory region of a vector, suchas a promoter, when the expression control sequence controls orregulates the transcription and/or the translation of that nucleic acidsequence. A nucleic acid that is “operably linked” to an expressioncontrol sequence includes, for example, an appropriate start signal(e.g., ATG) at the beginning of the nucleic acid sequence to beexpressed and a reading frame that permits expression of the nucleicacid sequence under control of the expression control sequence to yieldproduction of the encoded peptide. The regulatory region of theexpression vector optionally includes a termination sequence, such as acodon for which there is no corresponding aminoacetyl-tRNA, thus endingpeptide synthesis. Typically, when the ribosome reaches a terminationsequence or codon during translation of the mRNA, the polypeptide isreleased and the ribosome-mRNA-tRNA complex dissociates.

[0042] An expression vector optionally includes one or more selection ormarker sequences, which typically encode an enzyme capable ofinactivating a compound in the growth medium. The inclusion of a markersequence can, for example, render the host cell resistant to anantibiotic, or it can confer a compound-specific metabolic advantage onthe host cell. Markers such as green fluorescent protein also can beused to monitor growth or toxicity in host cells in which it isexpressed. Cells can be transformed with the expression vector using anyconvenient method known in the art, including chemical transformation,e.g., whereby cells are made competent by treatment with reagents suchas CaCl₂; electroporation and other electrical techniques;microinjection and the like.

[0043] The vector may further include a tightly regulable expressioncontrol sequence operably linked to the nucleic acid sequence encodingthe polypeptide, particularly a stabilized polypeptide, as describedherein. In embodiments of the method that use a tightly regulableexpression system derived from the lac operon, the host cell is or hasbeen genetically engineered or otherwise altered to contain a source ofLac repressor protein in excess of the amount produced in wild-type E.coli. A host cell that contains an excess source of Lac repressorprotein is one that expresses an amount of Lac repressor proteinsufficient to repress expression of the peptide under repressedconditions, i.e., in the absence of an inducing agent, such as isopropylβ-D-thiogalactoside (IPTG). Preferably, expression of Lac repressorprotein is constitutive. For example, the host cell can be transformedwith a second vector comprising a gene encoding Lac repressor protein,preferably lacI, more preferably lacIq, to provide an excess source ofLac repressor protein in trans, i.e., extraneous to the tightlyregulable expression vector. An episome can also serve as a trans sourceof Lac repressor. Another option for providing a trans source of Lacrepressor protein is the host chromosome itself, which can begenetically engineered to express excess Lac repressor protein.Alternatively, a gene encoding Lac repressor protein can be included onthe tightly regulable expression vector that contains thepeptide-encoding oligonucleotide so that Lac repressor protein isprovided in cis. The gene encoding the Lac repressor protein ispreferably under the control of a constitutive promoter.

[0044] The invention is not limited by the type of host cell used forscreening. The host cell can be a prokaryotic or a eukaryotic cell.Preferred mammalian cells include human cells, of any tissue type, andcan include cancer cells or cell lines (e.g., HeLa cells) or otherimmortalized cell lines, hybridomas, pluripotent or omnipotent cellssuch as stem cells or cord blood cells, etc., without limitation.Preferred yeast host cells include Saccharomyces cerevisiae andSchizosaccharomyces pombe. Preferred bacterial host cells include gramnegative bacteria, such as E. coli and various Salmonella spp., and grampositive bacteria, such as bacteria from the genera Staphylococcus,Streptococcus and Enterococcus. Protozoan cells are also suitable hostcells. In clear contrast to conventional recombinant protein expressionsystems, it is preferable that the host cell contains proteases and/orpeptidases, since the selection will, as a result, be advantageouslybiased in favor of peptides that are protease-and peptidase-resistant.More preferably, the host cell has not been modified, genetically orotherwise, to reduce or eliminate the expression of any naturallyexpressed proteases or peptidases. The host cell can be selected with aparticular purpose in mind. For example, if it is desired to obtainpeptide drugs specific to inhibit Staphylococcus, peptides can beadvantageously expressed and screened in Staphylococcus.

[0045] There is, accordingly, tremendous potential for the applicationof this technology in the development of new antibacterial peptidesuseful to treat various pathogenic bacteria. Of particular interest arepathogenic Staphylococci, Streptococci, and Enterococci, which are theprimary causes of nosocomial infections. Many of these strains arebecoming increasingly drug-resistant at an alarming rate. The technologyof the present invention can be practiced in a pathogenic host cell toisolate inhibitor peptides that specifically target the pathogenicstrain of choice. Inhibitory peptides identified using pathogenicmicrobial host cells in accordance with the invention may have directtherapeutic utility; based on what is known about peptide import, it isvery likely that small peptides are rapidly taken up by Staphylococci,Streptococci, and Enterococci. Once internalized, the inhibitorypeptides identified according to the invention would be expected toinhibit the growth of the bacteria in question. It is thereforecontemplated that novel inhibitor peptides so identified can be used inmedical treatments and therapies directed against microbial infection.It is further contemplated that these novel inhibitor peptides can beused, in turn, to identify additional novel antibacterial peptides usinga synthetic approach. The coding sequence of the inhibitory peptides isdetermined, and peptides are then chemically synthesized and tested inthe host cell for their inhibitory properties.

[0046] Novel inhibitor peptides identified in a pathogenic microbialhost cell according to the invention can also be used to elucidatepotential new drug targets. The protein target that the inhibitorpeptide inactivated is identified using reverse genetics by isolatingmutants that are no longer inhibited by the peptide. These mutants arethen mapped in order to precisely determine the protein target that isinhibited. New antibacterial drugs can then be developed using variousknown or yet to be discovered pharmaceutical strategies.

[0047] Following transformation of the host cell, the transformed hostcell is initially grown under conditions that repress expression of thepeptide. Expression of the peptide is then induced. For example, when alac promoter/operator system is used for expression, IPTG is added tothe culture medium. A determination is subsequently made as to whetherthe peptide is inhibitory to host cell growth, wherein inhibition ofhost cell growth under induced but not repressed conditions isindicative of the expression of a bioactive peptide.

[0048] Alternatively, a vector encoding a marker such as greenfluorescent protein (GFP) can be used to monitor toxicity of the randompeptides in a host. In general, fluorescence can be monitored in cellsthat are expressing both GFP and a randomized peptide and compared withthe fluorescence of control cells, i.e., cells expressing only GFP. Ifthe randomized peptide is toxic to the host, fluorescence would not beobserved or would be decreased relative to the control cells.Alternatively, GFP or other markers can be used to monitor the cells forcomplete or partial inhibition of cell division, or for induction ofapoptosis.

[0049] For example, to identify a potential anticancer peptide, a cancercell line such as the HeLa cell line can be used as the host. The cellline can be transfected with one or more vectors such that the cell lineexpresses both a marker (e.g., GFP) and a peptide from a library ofrandom peptides. It should be noted that the nucleic acid sequencesencoding the marker and the random peptides can be on the same ordifferent vectors. Expression of the random peptides can be controlledby a tightly regulable control sequence, although this need not be thecase. The transfected cells can be seeded into multi-well plates (e.g.,96-well plates) or into multiple flasks, with each well or platereceiving cells collectively expressing a single random peptide. In oneembodiment, the number of cells seeded into each well or flask is chosento ensure that an adequate number of cells expressing a random peptideand a marker is present in each well or flask. This can depend, forexample, on the original transfection efficiency. Under theseconditions, the marker will be observed in all wells or in each flask,unless a peptide that is being expressed is toxic to the cells orotherwise exhibits a desirable bioactivity (e.g., causes a complete orpartial inhibition of cell division, or induces apoptosis). In wells inwhich fluorescence is not observed or the level of fluorescence isdecreased, the random peptides are candidates for anti-cancer peptides.Candidate anti-cancer peptides identified by this method can be furtherscreened to determine if the peptide is selectively toxic or otherwisebioactive in cancer cells. For example, the bioactivity can be comparedbetween malignant and non-malignant cells using a 96-well screeningformat similar to that described above.

[0050] In a similar fashion, the method of the invention can be used toidentify peptides that exert an agonist effect on cell division andgrowth. For example, stem cells and cord blood cells, which typically donot proliferate well, can be employed as the host cells. Candidatepeptides can be assayed for a positive effect on cell division andgrowth. Agonistic peptides may be useful in wound healing, organtransplantation and cardiovascular applications.

[0051] A plurality of vectors (e.g., a library) that encode a populationof randomized peptides can be used to identify bioactive peptides (e.g.,antimicrobial or anticancer peptides). A library can include at leasttwo different vectors (e.g., at least five, 50, 500, 5000, 50,000,100,000, 1×10⁶, 5×10⁶ or more vectors), with each of the vectorsencoding different, randomized peptides (e.g., at least five, 50, 500,5000, 50,000, 100,000, 1×10⁶, 5×10⁶ or more different randomizedpeptides). Bioactive peptides can be identified by screening each of therandomized peptides encoded by the different vectors, for a desiredbioactivity (e.g., cell toxicity). Randomized peptides that exhibit thedesired bioactivity can be selected as bioactive peptides.

[0052] During development and testing of the intracellular screeningmethod of the present invention, it was surprisingly discovered thatseveral bioactive peptides identified from the randomized peptidelibrary shared particular structural features. For example, adisproportionately high number of bioactive peptides identified usingthe intracellular screening method contained one or more prolineresidues at or near a peptide terminus. A disproportionate number alsocontained sequences predicted, using structure prediction algorithmswell-known in the art, to form secondary structures such as a helices orsheets; or a hydrophobic membrane spanning domain. Bioactive fusionproteins comprising the randomized peptide sequence fused to the Ropprotein, due to a deletion event in the expression vector, were alsoidentified.

[0053] Accordingly, randomized peptides used in the screening method ofthe invention can be optionally engineered according to the method ofthe invention in a biased synthesis to increase their stability bymaking one or both of the N-terminal or C-terminal ends more resistantto proteases and peptidases. For example, a vector can include a nucleicacid sequence encoding a stabilized polypeptide, wherein the stabilizedpolypeptide includes a randomized peptide and a stabilizing grouppositioned at the N- and/or C-terminus of the randomized peptide. Theresulting stabilized polypeptide includes the randomized peptide and thestabilizing group coupled to one or both of the randomized peptide'stermini. By “coupled to . . . one or both . . . termini” it is meantthat the randomized peptide is covalently linked, at one or both of itstermini, to the stabilizing group. The nucleic acid sequence thatencodes the randomized peptide in the expression vector or theexpression vector itself is preferably modified such that a firststabilizing group is positioned at the N-terminus of the peptide, and asecond stabilizing group is positioned at the C-terminus of the peptide.

[0054] Notably, the bioactive peptides identified according to themethod of the invention are, by reason of the method itself, stable inthe intracellular environment of the host cell. The method of theinvention thus preferably identifies bioactive peptides that areresistant to proteases and peptidases. Resistance to proteases andpeptidases can be evaluated by measuring peptide degradation when incontact with appropriate cell extracts (e.g., bacterial, yeast, or humancell extracts), employing methods well-known in the art. A bioactivepeptide, without stabilization, can be used as a control. For example,degradation of a stabilized, biotinylated peptide can be assessed byelectrophoresis through an SDS-polyacrylamide gel and Western blottingusing an avidin-horseradish peroxidase conjugate. Alternatively,resistance to proteases and peptidases can be evaluated by measuringpeptide degradation when in contact with purified peptidases and/orproteases (e.g., the Lon and Clp proteases from E. coli). A protease- orpeptidase-resistant peptide exhibits a longer half-life in the presenceof proteases or peptidases compared to a control peptide.

[0055] In should be noted that the stabilization of peptides (e.g.,polypeptides containing about 2 to about 50 amino acids) in accordancewith the present invention is an unexpected as peptides, unlikeproteins, are relatively unstable in physiological environments. Forexample, the half-life of most peptides in physiological environments isabout 2 minutes, whereas the half-life of most proteins in physiologicalenvironments is typically well in excess of 2 minutes and is oftenmeasured in hours or days. Proteins possess an inherent stability as aresult of complex intramolecular interactions wherein, due to “proteinfolding” sections of the polypeptide that are distant on the linearchain are close together in space resulting in tertiary and quaternarystructure. Peptides, on the other hand, typically possess, at most, oneor two secondary structural elements (e.g., α-helix, β-sheet or β-turn).Many peptides possess no apparent secondary structural elements at all.

[0056] Stabilizing groups are amino acid sequences that can range insize from a single amino acid to a polypeptide (>50 amino acids).Suitable stabilizing groups do not specifically bind to serum proteins(e.g., albumin) or immunoglobulins, and in many embodiments, are free ofdisulfide bonds. Thus, the stabilizing groups of the present inventiondirectly stabilize the peptides to which they are attached. Stabilizinggroups that do not elicit, or elicit only minimal (i.e., clinicallyacceptable), immune responses in subject mammals are particularlyuseful.

[0057] In one embodiment, the stabilizing group is a stable protein,preferably a small stable protein such as thioredoxin, glutathionesulfotransferase, maltose binding protein, glutathione reductase, or afour-helix bundle protein such as Rop protein, as described below,although no specific size limitation on the protein anchor is intended.

[0058] Proteins suitable for use as stabilizing groups can be eithernaturally occurring or non-naturally occurring. They can be isolatedfrom an endogenous source, chemically or enzymatically synthesized, orproduced using recombinant DNA technology. Proteins that areparticularly well-suited for use as stabilizing groups are those thatare relatively short in length and form very stable structures insolution. Proteins having molecular weights of less than about 70 kD(e.g., less than about 65, 60, 50, 40, 25, or 12 kD) are useful asstabilizing groups. For example, human serum albumin has a molecularweight of about 64 kD; E. coli thioredoxin has a molecular weight ofabout 11.7 kD; E. coli glutathione sulfotransferase has a molecularweight of about 22.9 kD; Rop from the ColEl replicon has a molecularweight of about 7.2 kD; and maltose binding protein (without its signalsequence) has a molecular weight of about 40.7 kD. The small size of theRop protein makes it especially useful as a stabilizing group, fusionpartner, or peptide “anchor”, in that it is less likely than largerproteins to interfere with the accessibility of the linked peptide, thuspreserving its bioactivity. Rop's highly ordered anti-parallelfour-helix bundle topology (after dimerization), slow unfolding kinetics(see, e.g., Betz et al, Biochemistry 36, 2450-2458 (1997)), and lack ofdisulfide bonds also contribute to its usefulness as a peptide anchoraccording to the invention. Other proteins with similar folding kineticsand/or thermodynamic stability (e.g., Rop has a midpoint temperature ofdenaturation, T_(m), of about 71° C., Steif et al., Biochemistry 32,3867-3876 (1993)) are also preferred peptide anchors.

[0059] Peptides or proteins having highly stable tertiary structures,such as a four-helix bundle topology as exemplified in Rop, areparticularly useful. Thus, in another embodiment of the screening methodof the invention, the expression vector encodes a stabilizing groupcomprising an α-helical moiety at the N-terminus, C-terminus, or both,of the randomized peptide. The resulting fusion protein is predicted tobe more stable than the randomized peptide itself in the hostintracellular environment. Suitable α-helical moieties can range from asingle α-helix to two, three, four, or five α-helix bundles.

[0060] Non-limiting examples of single α-helical moieties that can beused to stabilize a bioactive peptide include the following: a 17 aminoacid peptide based on the first α-helix of the α-helix/turn/α-helixpeptide of Fezoui et al., Proc. Natl. Acad. Sci. USA 91, 3675-3679(1994)(Asp-Trp-Leu-Lys-Ala-Arg-Val-Glu-Gln-Glu-Leu-Gln-Ala-Leu-Glu-Ala-Arg,SEQ ID NO:111); an 18 to 36 amino acid peptide containing only glutamicacid, lysine, and glutamine residues, such as (Glu-Lys-Gln)y where y is6 to 12, although no specific arrangement of the three amino acidswithin the repeating tripeptide is intended; a 20 amino acid peptidecontaining amino acids 14-33 of Neuropeptide Y(Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg,SEQ ID NO:112); a 21 amino acid peptide based on amino acids 88-108 ofhuman mannose binding protein(Ala-Ala-Ser-Glu-Arg-Lys-Ala-Leu-Gln-Thr-Glu-Met-Ala-Arg-Ee-Lys-Lys-Ala-Leu-Thr-Ala,SEQ ID NO:113); a 24 amino acid peptide based on amino acids 4-27 ofhelodermin(Ala-Ile-Phe-Thr-Glu-Glu-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala-Leu-Gln-Lys-Tyr-Leu-Ala-Ser-Ile-Leu,SEQ ID NO:114); and a 34 amino acid peptide based on amino acids 41-74of ribosomal protein L9(Pro-Ala-Asn-Leu-Lys-Ala-Leu-Glu-Ala-Gln-Lys-Gln-Lys-Glu-Gln-Arg-Gln-Ala-Ala-Glu-Glu-Leu-Ala-Asn-Ala-Lys-Lys-Leu-Lys-Glu-Gln-Leu-Glu-Lys,SEQ ID NO:115).

[0061] Non-limiting examples of two-helix bundles include two α-helicesconnected by a turn region (see, for example, the 38 amino acidα-helix/turn/α-helix peptide of Fezoui et al. (1994), supra); a 42 aminoacid peptide based on amino acids 512-553 of the adhesion modulationdomain (AMD) of α-catenin; a 64 amino acid peptide based on residues411-475 of α-catenin; and a 78 amino acid a peptide based on residues24-102 of seryl-tRNA synthetase from E. coli.

[0062] Non-limiting examples of three-helix bundles include a peptidebased on residues 410-504 of α-catenin, a (Gly-Pro-Pro-)₁₀ or(-Pro-Pro-Gly)₁₀ peptide, and an (Ala-Pro-Pro-)₁₀ or (-Pro-Pro-Ala)₁₀peptide.

[0063] Two-helix bundles may dimerize and form a four-helix bundle. Asindicated above, Rop, which is 63 amino acids in size, forms a two-helixbundle that automatically dimerizes to become a four-helix bundle. Otheruseful four-helix bundles include the 35 amino acid and 51 amino acidfour-helix bundle peptides of Betz et al. (1997) supra, and the 125amino acid AMD of the α-catenin protein (residues 509-633 of theα-catenin protein).

[0064] Where a small stable protein or an α-helical moiety, such as afour-helix bundle protein, is fused to the N-terminus, the randomizedpeptide can optionally be further stabilized by, for example, covalentlylinking one or more prolines, with or without a following undefinedamino acids (e.g., -Pro, -Pro-Pro, -Pro-Xaa_(n), -Pro-Pro-Xaa_(n), etc.)at the C-terminus of the peptide sequence, where n is 1 or 2; likewise,when the α-helical moiety is fused to the C-terminus, the randomizedpeptide can be further stabilized by, for example, covalently linkingone or more prolines, with or without a preceding undefined amino acid(e.g., Pro-, Pro-Pro-, Xaa_(n)-Pro-, Xaa_(n)-Pro-Pro-, etc.) at theN-terminus of the peptide sequence, where n is 1 or 2, as discussed inmore detail below.

[0065] In another embodiment of the screening method of the invention,the stabilizing group can constitute one or more proline (Pro) residues.Preferably, a proline dipeptide (Pro-Pro) is used as a stabilizinggroup, although additional prolines may be included. The encodedproline(s) are typically naturally occurring amino acids. However, ifand to the extent a proline derivative, for example a hydroxyproline ora methyl- or ethyl-proline derivative, can be encoded or otherwiseincorporated into the peptide, those proline derivatives are also usefulas stabilizing groups.

[0066] At the N-terminus of the peptide, the stabilizing group also canbe an oligopeptide having the sequence Xaa_(n)-Pro_(m)-, wherein Xaa isany amino acid (e.g., Ala), m is greater than 0, and n is 1 or 2. Forexample, m can be about 1 to about 5 (e.g., m can be 2 or 3). Anoligopeptide having the sequence Xaa_(n)-Pro_(m)-, wherein m=2, isparticularly useful. Likewise, at the C-terminus of the peptide, thestabilizing group can be an oligopeptide having the sequence-Pro_(m)-Xaa_(n), wherein Xaa is any amino acid (e.g., Ala), m isgreater than 0, and n is 1 or 2. For example, m can be about 1 to about5 (e.g., m can be 2 or 3). An oligopeptide having the sequencePro_(m)-Xaa_(n), wherein m=2, is particularly useful.

[0067] In one embodiment of the screening method of the invention, thenucleic acid sequence that encodes the randomized peptide in theexpression vector is modified to encode both a first stabilizing grouplinked to the N-terminus of the peptide, the first stabilizing groupbeing selected from the group consisting of small stable protein, Pro-,Pro-Pro-, Xaa_(n)-Pro-, and Xaa_(n)-Pro-Pro-, and a second stabilizinggroup linked to the C-terminus of the peptide, the second stabilizinggroup being selected from the group consisting of a small stableprotein, -Pro, -Pro-Pro, Pro-Xaa_(n) and Pro-Pro-Xaa_(n). The resultingpeptide has enhanced stability in the intracellular environment relativeto a peptide lacking the terminal stabilizing groups.

[0068] In yet another embodiment of the screening method of theinvention, the putative bioactive peptide is stabilized by engineeringinto the peptide a stabilizing motif such as an α-helix motif or anopposite charge ending motif. Chemical synthesis of an oligonucleotideaccording to the scheme [(CAG)A(TCAG)] yields an oligonucleotideencoding a peptide consisting of a random mixture of the hydrophilicamino acids His, Gin, Asn, Lys, Asp, and Glu (see Table 14). Except foraspartate, these amino acids are most often associated with α-helicalsecondary structural motifs; the resulting oligonucleotides are thusbiased in favor of oligonucleotides that encode peptides that are likelyto form α-helices in solution.

[0069] Alternatively, the putative bioactive peptide is stabilized byflanking a randomized region with a region of uniform charge (e.g.,positive charge) on one end and a region of opposite charge (e.g.,negative) on the other end, to form an opposite charge-ending motif. Tothis end, the nucleic acid sequence that encodes the randomized peptidein the expression vector or the expression vector itself is preferablymodified to encode a plurality of sequential uniformly charged aminoacids at the N-terminus of the peptide, and a plurality of sequentialoppositely charged amino acids at the C-terminus of the peptide. Thepositive charges are supplied by a plurality of positively charged aminoacids consisting of lysine, histidine, arginine or a combinationthereof; and the negative charges are supplied by a plurality ofnegatively charged amino acids consisting of aspartate, glutamate or acombination thereof. It is expected that such a peptide will bestabilized by the ionic interaction of the two oppositely charged ends.Preferably, the putative bioactive peptide contains at least threecharged amino acids at each end. More preferably, it contains at leastfour charged amino acids at each end. In a particularly preferredembodiment, the larger acidic amino acid glutamate is paired with thesmaller basic amino acid lysine, and the smaller acidic amino acidaspartate is paired with the larger basic amino acid arginine.

[0070] The present invention further provides a bioactive peptidecontaining one or more structural features or motifs selected to enhancethe stability of the bioactive peptide in an intracellular environment.For example, a bioactive peptide of the invention can include anystabilizing group as described above in connection with the screeningmethod of the invention. Thus, stabilized bioactive peptides identifiedusing the screening method of the invention are included in theinvention. Likewise, both known bioactive peptides and bioactivepeptides subsequently discovered, when linked to one or more stabilizinggroups as described herein, are also within the scope of the presentinvention.

[0071] Accordingly, the invention provides a bioactive peptide having astabilizing group at its N-terminus, its C-terminus, or at both termini.

[0072] The bioactive peptide of the invention includes a bioactivepeptide that has been detectably labeled, derivatized, or modified inany manner desired prior to use, provided it contains one or moreterminal stabilizing groups as provided herein. For example, anon-stabilizing moiety (e.g., a label) can be attached to eitherterminus of the bioactive peptide, which terminus may or may not alsoinclude a stabilizing group.

[0073] The stabilized bioactive peptide of the invention can besynthesized enzymatically, chemically, or produced by recombinantgenetic engineering, without limitation, as described in more detailbelow. In any synthetic peptide having a stabilizing group that includesone or more prolines according to the present invention, the proline ispreferably a naturally occurring amino acid; alternatively, however, itcan be a synthetic derivative of proline, for example a hydroxyprolineor a methyl- or ethyl-proline derivative. Accordingly, where theabbreviation “Pro” is used herein in connection with a stabilizing groupthat is part of a synthetic peptide, it is meant to include prolinederivatives in addition to a naturally occurring proline.

[0074] In a bioactive peptide stabilized at only one terminus (i.e., ateither the N- or the C-terminus), the stabilizing group is preferably anα-helical moiety (e.g., four-helix bundle protein such as Rop protein),or one or more proline residues, with or without an undefined amino acid(Xaa). The resulting polypeptide consists essentially of a bioactivepeptide and the stabilizing group coupled to one terminus of thebioactive peptide.

[0075] A peptide stabilized at both termini can include a firststabilizing group attached to the N-terminus, and a second stabilizinggroup attached to the C-terminus, where the first and second stabilizinggroups are as defined previously in connection with the method foridentifying bioactive peptides. The stabilizing group is covalentlyattached to the peptide (e.g., via a peptide bond).

[0076] In one embodiment of the bioactive peptide of the invention, thefirst stabilizing group is Xaa_(n)-Pro_(m)-, with or without a precedingundefined amino acid (e.g., Pro-, Pro-Pro-, Xaa_(n)-Pro-,Xaa_(n)-Pro-Pro-, etc.), and the second stabilizing group is-Pro_(m)-Xaa_(n), with or without a following undefined amino acids(e.g., -Pro, -Pro-Pro, -Pro-Xaa_(n), -Pro-Pro-Xaa_(n), etc.). In anotherembodiment, the first (N-terminal) stabilizing group is a small stableprotein or an α-helical moiety (e.g., a four-helix bundle protein suchas Rop protein); and the second (C-terminal) stabilizing group is-Pro_(m)-Xaa_(n) or one or more proline residues (e.g., -Pro-Pro). Inyet another embodiment, the second (C-terminal) stabilizing group is asmall stable protein or an α-helical moiety (e.g., a four-helix bundleprotein such as Rop protein) and the first (N-terminal) stabilizinggroup is Xaa_(n)-Pro_(m)- or one or more proline residues.

[0077] The invention further provides a peptide stabilized by flankingthe amino acid sequence of a bioactive peptide with an opposite chargeending motif, as described herein. Preferably, the resulting stabilizedpeptide retains at least a portion of the biological activity of thebioactive protein. The stabilized peptide includes a peptide that hasbeen detectably labeled, derivatized, or modified in any manner desiredprior to use.

[0078] It should be understood that any bioactive peptide, withoutlimitation, can be stabilized according to the invention by attaching astabilizing group to either or both of the N- and C-termini. Included inthe present invention are various antimicrobial peptides, inhibitorypeptides, therapeutic peptide drugs, and the like. Non-limiting examplesinclude adrenocorticotropic hormone,bactericidal/permeability-increasing protein (BPI), brain natriureticpeptide, cercropin, endothelin, pentagastrin, scorpion peptides,teriparatide acetate, and all of the peptides listed in Tables 1 and 2,that have been modified at one or both peptide termini to include astabilizing group as discussed above. Particularly useful bioactivepeptides include insulin, glucagon, calcitonin, somatostatin,gonadotrophin, and secretin.

[0079] The invention is exemplified by peptides such asPro-Pro-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Pro-Pro (SEQ ID NO:3)andGlu-Asp-Glu-Asp-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-Arg-Lys-Arg-Lys(SEQ ID NO:4), wherein the middle nine amino acids(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Ile-; SEQ ID NO:5) constitute thesequence of angiotensin. In embodiments in which the bioactive peptideis a known peptide (e.g., angiotensin), the stabilizing group or groupsthat are coupled to one or both of the bioactive peptide's termini arenot naturally associated with the peptide. In other words, thestabilizing groups that are coupled to the bioactive peptide areheterologous to the bioactive peptide.

[0080] In embodiments in which a first stabilizing group is coupled tothe N-terminus of a bioactive peptide and a second stabilizing group iscoupled to the C-terminus of the bioactive peptide, the first and secondstabilizing groups can be the same or different. In some embodimentswhere the first and second stabilizing groups are different, they can besaid to be “heterologous” to each other, i.e., the stabilizing groupshave different amino acid sequences and (1) are from different proteins,or (2) are from the same protein, but the stabilizing groups are notcontiguous with each other in a naturally occurring protein, or (3) areproduced synthetically and one or both of the stabilizing groups do notcorrespond to a naturally occurring sequence. For example, a bioactivepeptide can be coupled to Rop at the N-terminus and coupled to atwo-helix bundle from the α-catenin AMD protein on the C-terminus.

[0081] Where the first and second stabilizing groups are from the sameprotein, it is not necessary that these flanking groups interact witheach other so as to confine or constrain the N-terminus and C-terminusof the flanked peptide in close proximity to one another. For example,Lavallie and others (Bio/Technology 11: 187-193 (1993); McCoy et al.,U.S. Pat. Nos. 5,270,181; 5,292,646; 5,646,016 and 6,143,524) describeinternal peptide fusions at the active site loop of thioredoxin, citingthe advantages of strong secondary structure in that region, the absenceof tertiary structure, and constraint of the peptide at both ends. Inthe internal thioredoxin fusion, the inserted peptide is bound at eachend by cysteine residues, which may form a disulfide linkage and furtherlimit the conformational freedom of the inserted peptide.

[0082] However, the present work suggests that neither steric constraintof the peptide ends nor any of the other unique characteristics ofthioredoxin polypeptides are necessary. Proteins other than thioredoxin,such as Rop, can be effectively used as first and/or second stabilizinggroups for bioactive peptides. Neither the first nor the secondstabilizing group needs to possess the capacity to participate in theformation of an intramolecular disulfide bond. That is, the stabilizinggroup does not need to contain a cysteine or, if it does, the remainderof the polypeptide need not contain a cysteine. The stabilizing groupscan be selected such that disulfide formation between the first andsecond stabilizing groups, if it occurs at all, does not bring theN-terminus and C-terminus of the bioactive peptide into close proximity.For example, flanking stabilizing groups can be selected such that theydo not contain, within about three residues of the ends that areconnected to the ends of the bioactive peptide, cysteine residues thatinteract with each other to form an intramolecular disulfide bond.

[0083] Moreover, in embodiments in which the first and secondstabilizing groups are from the same polypeptide, the stabilizing groupsneed not, although they may, interact to form a naturally occurringsecondary or tertiary structure. Naturally occurring secondarystructures include, for example, α-helices, β-sheets, β-turns and thelike that are present in, for example, the native solution or crystalstructure of the protein as determined by X-ray crystallography ornuclear magnetic resonance spectroscopy. Naturally occurring tertiarystructures result from the “folding” of sections the polypeptide thatmay be distant on the linear chain such that they are close together inspace. Tertiary structure includes the three-dimensional relationshipsbetween and among the secondary structures and unstructured portions ofthe molecule.

[0084] Modification of a bioactive peptide to yield a stabilizedbioactive peptide according to the invention can be achieved by standardtechniques well-known in the arts of genetics and peptide synthesis. Forexample, where the peptide is synthesized de novo, as in solid statepeptide synthesis, one or more prolines or other stabilizing groups canbe added at the beginning and the end of the peptide chain during thesynthetic reaction. In recombinant synthesis, for example as describedin Example III herein, one or more codons encoding proline, or codonsencoding α-helical moieties, for example, can be inserted into thepeptide coding sequence at the beginning and/or the end of the sequence,as desired. Preferably, codons encoding N-terminal prolines are insertedafter (i.e., 3′ to) the initiation site ATG (which encodes methionine).Analogous techniques are used to synthesize bioactive peptides having anopposite charge ending motif. When a known bioactive peptide is modifiedto yield a stabilized bioactive peptide according to the invention, theunmodified peptide can conveniently be used as a control in a protease-or peptidase-resistance assay as described hereinabove to confirm, ifdesired, that the modified peptide exhibits increased stability.

[0085] A stabilized bioactive peptide according to the invention caninclude a peptide whose bioactivity is evident from or identified in a“phage display” experiment. In “phage display” peptides are displayed onthe surface of phage and assayed for bioactivity. Displayed peptides aretethered at the one terminus, typically the C-terminus, to thebacteriophage surface. Their other terminus, typically the N-terminus,is free (i.e., non-fused). Structurally, the polypeptide produced in aphage display system is typically a fusion polypeptide that contains apeptide of interest at the N-terminus, followed by a phage protein atthe C-terminus. In some phage display polypeptides, however, the orderis reversed and the phage protein is at the N-terminus of thepolypeptide and the peptide of interest is at the C-terminus. The phageprotein is selected such that the polypeptide is displayed on thesurface of the bacteriophage. Examples include bacteriophage proteinspII and pVIII.

[0086] Phage display can be used to screen peptide libraries andidentify novel bioactive peptides. Bioactive peptides that are activewhen displayed typically continue to exhibit bioactivity when fabricatedsynthetically (i.e., without fusion to the phage protein), but theyfrequently exhibit instability in vivo. This may be due to the fact thatthe C-terminus is no longer protected or tethered. Hence, the presentinvention includes a method for stabilizing a bioactive peptide bylinking a stabilizing group to the N-terminus or C-terminus of apeptide, when the bioactive peptide has been identified using phagedisplay.

[0087] Additionally or alternatively, the genetic constructs used toproduce the fusion protein within the bacteriophage can be engineeredencode a stabilizing group at the terminus of encoded fusion polypeptidethat would otherwise have been free, such that the fusion polypeptidedisplayed on the surface of the bacteriophage contains a peptide ofinterest flanked by a stabilizing group at the terminus and abacteriophage protein at the other terminus.

[0088] The invention thus includes methods for phage display ofstabilized bioactive proteins, methods for stabilizing bioactivepeptides identified using phage display, and bioactive peptides thusidentified and/or stabilized.

[0089] The present invention also provides a cleavable polypeptidecomprising a stabilized, bioactive peptide either immediately precededby (i.e., adjacent to the N-terminus of the bioactive peptide) acleavage site, or immediately followed by (i.e., adjacent to theC-terminus of the bioactive peptide) a cleavage site. Thus, a bioactivepeptide as contemplated by the invention can be part of a cleavablepolypeptide. The cleavable polypeptide is cleavable, either chemically,as with cyanogen bromide, or enzymatically, to yield the bioactivepeptide. The resulting bioactive peptide either includes a firststabilizing group positioned at its N-terminus and/or a secondstabilizing group positioned at its C-terminus, both as describedhereinabove. The cleavage site immediately precedes the N-terminalstabilizing group or immediately follows the C-terminal stabilizinggroup. In the case of a bioactive peptide stabilized with an oppositecharge ending motif, the cleavage site immediately precedes the firstcharged region or immediately follows the second charged region. Thecleavage site makes it possible to administer a bioactive peptide in aform that could allow intracellular targeting and/or activation.

[0090] Alternatively, a bioactive peptide of the invention can be fusedto a noncleavable N-terminal or C-terminal targeting sequence whereinthe targeting sequence allows targeted delivery of the bioactivepeptide, e.g., intracellular targeting or tissue-specific targeting ofthe bioactive peptide. In one embodiment of this aspect of theinvention, a stabilizing group (e.g., one or more proline residues) ispositioned at the free (i.e., non-fused) terminus of the bioactivepeptide as described hereinabove in connection with the screening methodfor identifying bioactive peptides. The targeting sequence attached tothe other peptide terminus can, but need not, contain a small stableprotein such as Rop or one or more proline residues, as long as thetargeting function of the targeting sequence is preserved. In anotherembodiment of this aspect of the invention, the bioactive peptide isstabilized with a charge ending motif as described hereinabove, whereinone charged region is coupled to the free terminus of the bioactivepeptide, and the other charged region is disposed between the targetingsequence and the active sequence of the bioactive peptide.

[0091] The invention further includes a method for using anantimicrobial peptide that includes covalently linking a stabilizinggroup, as described above, to the N-terminus, the C-terminus, or to bothtermini, to yield a stabilized antimicrobial peptide, then contacting amicrobe with the stabilized antimicrobial peptide. Alternatively, thestabilized antimicrobial peptide used in this aspect of the invention ismade by covalently linking oppositely charged regions, as describedabove, to each end of the antimicrobial peptide to form an oppositecharge ending motif. An antimicrobial peptide is to be broadlyunderstood as including any bioactive peptide that adversely affects amicrobe such as a bacterium, virus, protozoan, or the like, as describedin more detail above. An example of an antimicrobial peptide is aninhibitory peptide that inhibits the growth of a microbe. When theantimicrobial peptide is covalently linked to a stabilizing group atonly one peptide terminus, any of the stabilizing groups describedhereinabove can be utilized. When the antimicrobial peptide iscovalently linked to a stabilizing group at both peptide termini, themethod includes covalently linking a first stabilizing group to the Nterminus of the antimicrobial peptide and a second stabilizing group tothe C terminus of the antimicrobial peptide, where the first and secondstabilizing groups are as defined previously in connection with themethod for identifying bioactive peptides. In a preferred embodiment ofthe method for using an antimicrobial peptide, one or more prolines,more preferably a Pro-Pro dipeptide, is attached to at least one,preferably both, termini of the antimicrobial peptide. Alternatively, orin addition, an Xaa_(n)-Pro_(m)-sequence, as described above, can beattached to the N-terminus of a microbial peptide, and/or a-Pro_(m)-Xaa_(n) sequence can be attached to the C-terminus, to yield astabilized antimicrobial peptide.

[0092] The antimicrobial peptide thus modified in accordance with theinvention has enhanced stability in the intracellular environmentrelative to an unmodified antimicrobial peptide. As noted earlier, theunmodified peptide can conveniently be used as a control in a protease-or peptidase-resistance assay as described hereinabove to confirm, ifdesired, that the modified peptide exhibits increased stability.Further, the antimicrobial activity of the antimicrobial peptide ispreferably preserved or enhanced in the modified antimicrobial peptide;modifications that reduce or eliminate the antimicrobial activity of theantimicrobial peptide are easily detected and are to be avoided.

[0093] The invention further provides a method for inhibiting the growthof a microbe comprising contacting the microbe with a stabilizedinhibitory peptide. As described above, the stabilized inhibitorypeptide can have a stabilizing group attached at its N-terminus,C-terminus, or both termini.

[0094] Also included in the present invention is a method for treating apatient having a condition treatable with a peptide drug, comprisingadministering to the patient a peptide drug that has been stabilized asdescribed herein. Peptide drugs for use in therapeutic treatments arewell known (see, e.g., Table 1). However, they are often easily degradedin biological systems, which affects their efficacy. In one embodimentof the present method, the patient is treated with a stabilized drugcomprising the peptide drug of choice and a stabilizing group linked toeither the N-terminus, the C-terminus of, or to both termini of thepeptide drug. In another embodiment of the present method, the patientis treated with a stabilized drug comprising the peptide drug of choicethat has been stabilized by attachment of oppositely charged regions toboth termini of the peptide drug. Because the peptide drug is therebystabilized against proteolytic degradation, greater amounts of the drugshould reach the intended target in the patient.

[0095] In embodiments of the method involving administration of apeptide drug that is covalently linked to a stabilizing group at onlyone peptide terminus, the stabilizing group is preferably an α-helicalmoiety, such as a four-helix bundle protein (e.g., Rop), provided thatattachment of the α-helical moiety to the peptide terminus preserves asufficient amount of efficacy for the drug. It is to be nonethelessunderstood that the group or groups used to stabilize the peptide drugare as defined hereinabove, without limitation. In embodiments involvingadministration of a peptide drug covalently linked to a stabilizinggroup at both peptide termini, the peptide drug includes a firststabilizing group linked to the N-terminus of the peptide drug and asecond stabilizing group linked to the C-terminus of the peptide drug.Thus, in another preferred embodiment of the treatment method of theinvention, the stabilized peptide drug includes one or more prolines,more preferably a proline-proline dipeptide, attached to one or bothtermini of the peptide drug. For example, the peptide drug can bestabilized by covalent attachment of a Rop protein at one terminus, andby covalent attachment of a proline or proline dipeptide at the otherterminus; in another preferred embodiment, the peptide drug can bestabilized by proline dipeptides at each of the N-terminus and Cterminus. Alternatively, or in addition, the stabilized peptide drugused in the treatment method can include an Xaa_(n)-Pro_(m)-sequence atthe N-terminus of the peptide drug, and/or a -Pro_(m)-Xaa_(n) sequenceat the C-terminus. Optionally, prior to administering the stabilizedpeptide drug, the treatment method can include covalently linking astabilizing group to one or both termini of the peptide drug to yieldthe stabilized peptide drug.

[0096] If desired, the unmodified peptide drug can conveniently be usedas a control in a protease- or peptidase-resistance assay as describedhereinabove to confirm that the stabilized peptide drug exhibitsincreased stability. Further, the therapeutic efficacy of the peptidedrug is preferably preserved or enhanced in the stabilized peptide drug;modifications that reduce or eliminate the therapeutic efficacy of thepeptide drug are easily detected and are to be avoided.

[0097] The present invention further includes a fusion proteincomprising a four-helix bundle protein, preferably Rop protein, and apolypeptide. Preferably the polypeptide is bioactive; more preferably itis a bioactive peptide. The fusion protein of the invention can be usedin any convenient expression vector known in the art for expression oroverexpression of a peptide or protein of interest. Optionally, acleavage site is present between the four-helix bundle protein and thepolypeptide to allow cleavage, isolation and purification of thepolypeptide. In one embodiment of the fusion protein, the four-helixbundle protein is covalently linked at its C-terminus to the N-terminusof the polypeptide; in an alternative embodiment, the four-helix bundleprotein is covalently linked at its N-terminus to the C-terminus of thepolypeptide. Fusion proteins of the invention, and expression vectorscomprising nucleic acid sequences encoding fusion proteins wherein thenucleic acid sequences are operably linked to a regulatory controlelement such as a promoter, are useful for producing or overproducingany peptide or protein of interest.

EXAMPLES

[0098] The present invention is illustrated by the following examples.It is to be understood that the particular examples, materials, amounts,and procedures are to be interpreted broadly in accordance with thescope and spirit of the invention as set forth herein.

Example I Construction and Characterization of a Highly RegulableExpression Vector, pLAC11, and its Multipurpose Derivatives, pLAC22 andpLAC33

[0099] A number of different expression vectors have been developed overthe years to facilitate the production of proteins in E. coli andrelated bacteria. Most of the routinely employed expression vectors relyon lac control in order to overproduce a gene of choice. While thesevectors allow for overexpression of the gene product of interest, theyare leaky due to changes that have been introduced into the lac controlregion and gene expression can never be shut off under repressedconditions, as described in more detail below. Numerous researchers havenoticed this problem with the more popular expression vectors pKK223-3(G. Posfai et al. Gene. 50: 63-67 (1986); N. Scrutton et al., Biochem J.245: 875-880 (1987)), pKK233-2 (P. Beremand et al., Arch BiochemBiophys. 256: 90-100 (1987); K. Ooki et al., Biochemie. 76: 398-403(1994)), pTrc99A (S. Ghosh, Protein Expr. Purif. 10: 100-106 (1997); J.Ranie et al., Mol. Biochem. Parasitol. 61: 159-169 (1993)), as well asthe PET series (M. Eren et al., J. Biol. Chem. 264: 14874-14879 (1989);G. Godson, Gene 100: 59-64 (1991)).

[0100] The expression vector described in this example, pLAC11, wasdesigned to be more regulable and thus more tightly repressible whengrown under repressed conditions. This allows better regulation ofcloned genes in order to conduct physiological experiments. pLAC11 canbe used to conduct physiologically relevant studies in which the clonedgene is expressed at levels equal to that obtainable from thechromosomal copy of the gene in question. The expression vectorsdescribed here were designed utilizing the wild-type lacpromoter/operator in order to accomplish this purpose and include all ofthe lac control region, without modification, that is contained betweenthe start of the O3 auxiliary operator through the end of the O0operator. As with all lac based vectors, the pLAC11 expression vectordescribed herein can be turned on or off by the presence or absence ofthe gratuitous inducer IPTG. In experiments in which a bacterial cellcontained both a null allele in the chromosome and a second copy of thewild-type allele on pLAC11 cells grown under repressed conditionsexhibited the null phenotype while cells grown under induced conditionsexhibited the wild-type phenotype. Thus the pLAC11 vector truly allowsfor the gene of interest to be grown under either completely repressedor fully induced conditions. Two multipurpose derivatives of pLAC11,pLAC22 and pLAC33 were also constructed to fulfill differentexperimental needs.

[0101] The vectors pLAC11, pLAC22 and pLAC33 were deposited with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va., 20110-2209, USA, on Feb. 16, 1999, and assigned ATCCdeposit numbers ATCC 207108, ATCC 207110 and ATCC 207109, respectively.It is nonetheless to be understood that the written description hereinis considered sufficient to enable one skilled in the art to fullypractice the present invention. Moreover, the deposited embodiment isintended as a single illustration of one aspect of the invention and isnot to be construed as limiting the scope of the claims in any way.

[0102] Materials and Methods

[0103] Media. Minimal M9 media (6 g disodium phosphate, 3 g potassiumphosphate, 1 g ammonium chloride, 0.5 g sodium chloride, distilled waterto 1 L; autoclave; add 1 mL m magnesium sulfate (1M) and 0.1 mL calciumchloride (1M); a sugar added to a final concentration of 0.2%; vitaminsand amino acids as required for non-prototrophic strains) and rich LBmedia (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilledwater to 1 L; autoclave) were prepared as described by Miller (J.Miller, “Experiments in molecular genetics” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1972). The antibiotics ampicillin,kanamycin, streptomycin, and tetracycline (Sigma Chemical Company, St.Louis, Mo.) were used in rich media at a final concentration of 100, 40,200, and 20 ug/ml, respectively. When used in minimal media,tetracycline was added at a final concentration of 10 îg/ml.5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (Xgal) was added tomedia at a final concentration of 40 îg/ml unless otherwise noted. IPTGwas added to media at a final concentration of 1 mM.

[0104] Chemicals and Reagents. When amplified DNA was used to constructthe plasmids that were generated in this study, the PCR reaction wascarried out using native Pfu polymerase from Stratagene (Cat. No.600135). Xgal and IPTG were purchased from Diagnostic Chemicals Limited.

[0105] Bacterial Strains and Plasmids. Bacterial strains and plasmidsare listed in Table 4. To construct ALS225, ALS224 was mated with ALS216and streptomycin resistant, blue recombinants were selected on a Rich LBplat that contained streptomycin, Xgal, and IPTG. To construct ALS226,ALS224 was mated with ALS217 and streptomycin resistant, kanomycinresistant recombinants were selected on a Rich LB plate that containedstreptomycin and kanamycin. To construct ALSS15, ALSS14 was mated withALS216 and streptomycin resistant, blue recombinants were selected on aRich LB plate that contained streptomycin, Xgal, and IPTG. To constructALS527, ALS524 was mated with ALS224 and streptomycin resistant,tetracycline resistant recombinants were selected on a Rich LB platethat contained streptomycin and tetracycline. To construct ALS535,ALS533 was mated with ALS498 and tetracycline resistant recombinantswere selected on a Minimal M9 Glucose plate that contained tetracycline,leucine and thiamine (B₁) (Sigma Chemical Company). To construct ALS533,a P1 lysate prepared from E. coli strain K5076 (H. Miller et al., Cell20: 711-719 (1980)) was used to transduce ALS224 and tetracyclineresistant transductants were selected. TABLE 4 Bacterial strains andplasmids used in Example I E. coli Strains Laboratory Original Name NameGenotype Source ALS216 SE9100 araD139 Δ(lac)U169 thi E. Altman et al.,J. Biol. flbB5301 deoC7 ptsF25 Chem. 265: 18148-18153 rpsE/F′ lacl

¹ Z⁺ Y⁺ A⁺ (1990) ALS217 SE9100.1 araD139 Δ(lac)U169 thi S. Emr (Univ.of California, flbB5301 deoC7 ptsF25 San Diego) rpsE/F′ lacl

¹ Z::Tn5 Y+ A+ ALS221 BL21(DE3) ompT hsdS(b) (R-M-) gal F. Studier etal., J. Mol. Biol. dcm 189: 113-130 (1986) ALS224 MC1061 araD139Δ(araABOIC- M. Casadaban et al., J. Mol. leu) 7679 Δ(lac)X74 galU Biol.138: 179-207 (1980) galK rpsL hsr− hsm+ ALS225 MC1061/F′ lacl

¹ Z⁺ Y⁺ A⁺ This example ALS226 MC1061/F′ lacl

¹ Z::Tn5 This example Y⁺ A⁺ ALS269 CSH27 F- trpA33 thi J. Miller,“Experiments in molecular genetics” Cold Spring Laboratory, Cold SpringHarbor, NY (1972) ALS413 MG1655 E. coli wild-type F- λ- M. Guyer et al.,Cold Spring Harbor Symp. Quant. Biol. 45: 135-140 (1980) ALS498 JM101supE thi Δ(lac-proAB)/ C. Yanisch-Perron et al., F′ traD36 proA⁺B⁺ lacl

Gene. 33: 103-119 (1985) Δ(lacZ) M15 ALS514 NM554 MC1061 recA13 E.Raleigh et al., Nucl. Acids Res. 16: 1563-1575 (1988) ALS515 MC1061recA13/F′ This example lacl

¹ Z⁺ Y⁺ A⁺ ALS524 XL1-Blue recAI endAI gyrA96 thi-I Stratagene (Cat. No.200268) hsdRI7 supE44 relAI lac/F′ proAB lacl

Δ(lacZ) M15 Tn10 ALS527 MC1061/F′ proAB lacl

This example Δ (lacZ) M15 Tn10 ALS533 MC1061 proAB::Tn10 This exampleALS535 MC1061 proAB::Tn10/ This example F′ lacl

Δ(lacZ) M15 proA+B+ ALS598 CAG18615 zjb-3179::Tn10dKan M. Singer et al.,Microbiol. lambda-rph-1 Rev. 53: 1-24 (1989) Plasmids Plasmid NameRelevant Characteristics Source pBH20 wild-type lac promoter/ K. Itakuraet al., Science. 198: operator, Amp^(R), Tet^(R), colEl 1056-1063 (1977)replicon pBR322 Amp^(R), Tet^(R), colEl replicon F. Bolivar et al.,Gene. 2: 95-113 (1977) pET-21(+) T7 promoter/lac operator, laclq,Novagen (Cat. No. 69770-1) Amp^(R), colEl replicon pGE226 wild-type recAgene, Amp^(R) J. Weisemann, et al., J. Bacteriol. 163: 748-755 (1985)pKK223-3 tac promoter/operator, Amp^(R), J. Brosius et al., Proc. Natl.Acad. Sci. colEl replicon USA 81: 6929-6933 (1984) pKK223-2 trcpromoter/operator, Amp^(R), E. Amann et al., Gene. 40: 183-190 colElreplicon (1985) pLysE T7 lysozyme, Cam^(R), P15A F. Studier, J. Mol.Biol. 219: 37-44 replicon (1991) pLysS T7 lysozyme, Cam^(R), P15A F.Studier, J. Mol. Biol. 219: 37-44 replicon (1991) pMS421 wild-type lacpromoter/ D. Grana et al., Genetics. 120: 319-327 operator, laclq,Strep^(R), Spec^(R), (1988) SC101 replicon pTer7 wild-type lacZ codingregion, R. Young (Texas A&M University) Amp^(R) pTrc99A trcpromoter/operator, laclq, E. Amann et al., Gene. 69: 301-315 Amp^(R),colEl replicon (1988) pUC8 lac promoter/operator, Amp^(R), J. Vieira etal., Gene. 19: 259-268 colEl replicon (1982) pXE60 wild-type TOL pWWOxylE J. Westpheling (Univ. of Georgia) gene, Amp^(R)

[0106] Construction of the pLAC11, pLAC22, and pLAC33 expressionvectors. To construct pLAC11, primers #1 and #2 (see Table 5) were usedto PCR amplify a 952 base pair (bp fragment from the plasmid pBH20 whichcontains the wild-type lac operon. Primer #2 introduced two differentbase pair mutations into the seven base spacer region between the ShineDalgarno site and the ATG start site of the lacZ which converted it fromAACAGCT to AAGATCT thus placing a Bgl II site in between the ShineDalgarno and the start codon of the lacZ gene. The resulting fragmentwas gel isolated, digested with Pst I and EcoR I, and then ligated intothe 3614 bp fragment from the plasmid pBR322ΔAvaI which had beendigested with the same two restriction enzymes. To constructpBR322ΔAval, pBr322 was digested with Aval, filled in using Klenow, andthen religated. To construct pLAC22, a 1291 bp Nco I. EcoR I fragmentwas gel isolated from pLAC21 and ligated to a 4361 bp Nco I. EcoR Ifragment which was gel isolated from pBR322/NcoI. To construct pLAC21,primers #2 and #3 (see Table 5) were used to PCR amplify a 1310 bpfragment from the plasmid pMS421 which contains the wild-type lac operonas well as the lacIq repressor. The resulting fragment was gel isolated,digested with EcoR I, and then ligated into pBR322 which had also beendigested with EcoR I. To construct pBR322/Nco I, primers #4 and #5 (seeTable 5) were used to PCR amplify a 788 bp fragment from the plasmidpBR322. The resulting fragment was gel isolated, digested with Pst I andEcoR l, and then ligated into the 3606 bp fragment from the plasmidpBR322 which had been digested with the same two restriction enzymes.The pBR322Mco I vector also contains added Kpn 1 and Sma I sites inaddition to the new NcoI site. To construct pLAC33, a 2778 bp fragmentwas gel isolated from pLAC12 which had been digested with BsaB I and BsaI and ligated to a 960 bp fragment from pUC8 which had been digestedwith Afl III, filled in with Klenow, and then digested with Bsa I. Toconstruct pLAC12, a 1310 bp Pst I, BamH I fragment was gel isolated frompLAC11 and ligated to a 3232 bp Pst I, BamH I fragment which was gelisolated from pBR322. TABLE 5 Primers employed to PCR amplify DNAfragments that were used in the construction of the various plasmidsdescribed in Example 1 pLAC11 and pLAC22  1 (for) GTT GCC ATTGCT GCA GGC AT (SEQ ID NO:6)  2 (rev) ATT GAA TTC ATA AGA TCTTTC CTG TGTGAA ATT GTT ATC CGC (SEQ ID NO:7)  3 (for) ATT GAA TTC ACC ATG GAC ACCATC GAA TGG TGC AAA A (SEQ ID NO:8) pBR322/Nco I  4 (for) GTT GTT GCCATT GCT GCA G (SEQ ID NO:9)  5 (rev) TGT ATG AAT TCC CGG GTA CCA TGG TTGAAG ACG AAA GGG CCT C (SEQ ID NO:10) Bgl II-lacZ-Hind III  6 (for) TACTAT AGA TCT ATG ACC ATG ATT ACG GAT TCA CTG (SEQ ID NO:11)  7 (rev) TACATA AAG CTT GGC CTG CCC GGT TAT TAT TAT TTT (SEQ ID NO:12) PstI-lacZ-Hind III  8 (for) TAT CAT CTG CAG AGG AAA CAG CTA TGA CCA TGA TTACGG ATT CAC TG (SEQ ID NO:13)  9 (rev) TAC ATA CTC GAG CAG GAA AGC TTGGCC TGC CCG GTT ATT ATT ATT TT (SEQ ID NO:14) BamH 1-lacZ-Hind III (alsouses primer #9) 10 (for) TAT CAT GGA TCC AGG AAA CAG CTA TGA CCA TGA TTACGG ATT CAC TG (SEQ ID NO:15) Bgl II-recA-Hind III 11 (for) TAC TATAGA TCT ATG GCT ATC GAC GAA AAC AAA CAG (SEQ ID NO:16) 12 (rev) ATA TATAAG CTT TTA AAA ATC TTC GTT AGT TTC TGC TAC G (SEQ ID NO:17) BamH1-xylE-EcoR I 13 (for) TAC TAT AGA TCT ATG AAC AAA GGT GTA ATG CGA CC(SEQ ID NO:18) 14 (rev) ATT AGT GAA TTCGCA CAA TCT CTG CAA TAA GTC GT(SEQ ID NO:19)

[0107] In Table 5 the regions of the primers that are homologous to theDNA target template are indicated with a dotted underline, while therelevant restriction sites are indicated with a solid underline. Allprimers are listed in the 5′→3′ orientation.

[0108] Compilation of the DNA sequences for the pLAC11, pLAC22, andpLAC33 expression vectors. All of the DNA that is contained in thepLAC11, pLAC22, and, pLAC33 vectors has been sequenced.

[0109] The sequence for the pLAC11 vector, which is 4547 bp, can becompiled as follows: bp 1-15 are AGATCTTATGAATTC (SEQ ID NO: 20) fromprimer #2 (Table 5); bp 16-1434 are bp 4-1422 from pBR322 (GenBankAccession #JO1749); bp 1435-1442 are TCGGTCGG, caused by filling in theAva I site in pBR322AAvaI; bp 1443-4375 is bp 1427-4359 from pBR322(GenBank Accession #JO1749); and bp 4376-4547 are bp 1106-1277 from thewild-type E. coli lac operon (GenBank Accession #J01636).

[0110] The sequence for the pLAC22 vector which is 5652 bp can becompiled as follows: bp 1-15 are AGATCTTATGAA 'ITC (SEQ ID NO: 21) fromprimer #2 (Table 5); bp 16-4370 are bp 4-4358 from pBR322 (GenBankAccession# J01749); bp 4371-4376 is CCATGG which is the Nco I site frompBR322/Nco I; and bp 4377-5652 are bp 2-1277 from the wild-type E. colilac operon (GenBank Accession #J01636), except that bp #4391 of thepLAC22 sequence or bp#16 from the wild-type E. coli lac operon sequencehas been changed from a “C” to a “T” to reflect the presence of thelacIq mutation (J. Brosius et al., Proc. Natl. Acad. Sci. USA. 81:6929-6933 (1984)).

[0111] The sequence for the pLAC33 vector which is 3742 bp can becompiled as follows: bp 1-15 is AGATCTTATGAATTC (SEQ.ID NO: 22) fromprimer #2 (Table 5); bp 16-1684 are bp 4-1672 from pEk322 (GenBankAccession #501749); bp 1685-2638 are bp 786-1739 from pUC8 (GenBankAccession #L09132); bp 2639-3570 are bp 3428-4359 from pBR322 (GenBankAccession #30 J01749); and bp 3571-3742 are bp 1106-1277 from thewild-type E. coli lac operon (GenBank Accession #J01636). In the mapsfor these vectors, the ori is identified as per Balbás (P. Balbás etal., Gene. 50: 3-40 (1986)), while the lacPO is indicated starting withthe O3 auxiliary operatic and ending with the O1 operator as perMüller-Hill (B. Müller-Hill, The lac Operon: A Short History of aGenetic Paradigm. Walter de Gruyter, Berlin, Germany (1996)).

[0112] Construction of the pLAC11-, pLAC22-, pLAC33-, pKK223-3-,pKK233-2-, pTrc99A-, and pET-21(+)-lacZ constructs. To constructpLAC11-lacZ, pLAC22-lacZ, and pLAC33-lacZ, primers #6 and #7 (see Table5) were used to PCR amplify a 3115 bp fragment from the plasmid pTer7which contains the wild-type lacZ gene. The resulting fragment was gelisolated, digested with Bgl II and Hind III, and then ligated into thepLAC11, pLAC22 or pLAC33 vectors that had been digested with the sametwo restriction enzymes. To construct pKK223-3-lacZ and pKK233-2-lacZ,primers #8 and #9 (see Table 5) were used to PCR amplify a 3137 bpfragment from the plasmid pTer7. The resulting fragment was gelisolated, digested with Pst I and Hind III, and then ligated into thepKK223-3 or pKK233-2 vectors which had been digested with the same tworestriction enzymes. To construct pTrc99A-lacZ and pET-21(+)-lacZ,primers #9 and #10 (see Table 5) were used to PCR amplify a 3137 bpfragment from the plasmid pTer7. The resulting fragment was gelisolated, digested with BamH I and Hind III, and then ligated into thepTrc99A or pET-21 (+) vectors which had been digested with the same tworestriction enzymes.

[0113] Construction of the pLAC11-recA and xylE constructs. To constructpLAC11-recA, primers #11 and #12 (see Table 5) were used to PCR amplifya 1085 bp fragment from the plasmid pGE226 which contains the wild-typerecA gene. The resulting fragment was gel isolated, digested with Bgl IIand Hind III, and then ligated into the pLAC11 vector which had beendigested with the same two restriction enzymes. To constructpLAC11-xylE, primers #13 and #14 (see Table 5) were used to PCR amplifya 979 bp fragment from the plasmid pXE60 which contains the wild-typePseudomonas putida xylE gene isolated from the TOL pWWO plasmid. Theresulting fragment was gel isolated, digested with Bgl II and EcoR I,and then ligated into the pLAC11 vector which had been digested with thesame two restriction enzymes.

[0114] Assays. β-galactosidase assays were performed as described byMiller (J. Miller. “Experiments in molecular genetics,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1972)), while catechol2,3-dioxygenase (catO₂ase) assays were performed as described byZukowski, et. al. (M. Zukowski et al., Proc. Natl. Acad. Sci. U.S.A. 80:1101-1105 (1983)).

[0115] Results

[0116] Construction and features of pLAC11, pLAC22, and pLAC33. Plasmidmaps that indicate the unique restriction sites, drug resistances,origin of replication, and other relevant regions that are contained inpLAC11, pLAC22, and pLAC33 are shown in FIGS. 2, 3 and 4, respectively.pLAC11 was designed to be the most tightly regulable of these vectors.It utilizes the ColEl origin of replication from pBR322 and Laclrepressor is provided in trans from either an episome or anothercompatible plasmid. pLAC22 is very similar to pLAC11, however, it alsocontains lacIq, thus a source of LacI does not have to be provided intrans. pLAC33 is a derivative of pLAC11 which utilizes the mutated ColElorigin of replication from pUC8 (S. Lin-Chao et al., Mol. Micro. 6:3385-3393 (1992))and thus pLAC33's copy number is significantly higherthan pLAC11 and is comparable to that of other pUC vectors. Because thecloning regions of these three vectors are identical, cloned genes canbe trivially shuffled between and among them depending on the expressiondemands of the experiment in question.

[0117] To clone into pLAC11, pLAC22, or pLAC33, PCR amplification isperformed with primers that are designed to introduce unique restrictionsites just upstream and downstream of the gene of interest. Usually aBgl II site is introduced immediately in front of the ATG start codonand an EcoR I site is introduced immediately following the stop codon.An additional 6 bases is added to both ends of the oligonucleotide inorder to ensure that complete digestion of the amplified PCR productwill occur. After amplification the double-stranded (ds) DNA isrestricted with Bgl II and EcoR I, and cloned into the vector which hasalso been restricted with the same two enzymes. If the gene of interestcontains a BlgII site, then BamH I or Bcl I can be used instead sincethey generate overhangs which are compatible with Bgl II. If the gene ofinterest contains an EcoR I site, then a site downstream of EcoR I inthe vector (such as Hind III) can be substituted.

[0118] Comparison of pLAC11, pLAC22, and pLAC33, to other expressionvectors. In order to demonstrate how regulable the pLAC11, pLAC22, andpLAC33 expression vectors were, the wild-type lacZ gene was cloned intopLAC11, pLAC22, pLAC33, pKK223-3, pKK233-2, pTrc99A, and pET-21(+).Constructs which required an extraneous source of Lacl for theirrepression were transformed into ALS225, while constructs whichcontained a source of Lacl on the vector were transformed into ALS224.pET-21(+)constructs were transformed into BL21 because they require T7RNA polymerase for their expression. Four clones were chosen for each ofthese seven constructs and β-galactosidase assays were performed underrepressed and induced conditions. Rich Amp overnights were diluted 1 to200 in either Rich Amp Glucose or Rich Amp IPTG media and grown untilthey reached mid-log (OD₅₅₀=0.5). In the case of PET-21(+) the pLysE andpLysS plasmids, which make T7 lysozyme and thus lower the amount ofavailable T7 polymerase, were also transformed into each of theconstructs. Table 6 shows the results of these studies and also liststhe induction ratio that was determined for each of the expressionvectors. As the data clearly indicate, pLAC11 is the most regulable ofthese expression vectors and its induction ratio is close to that whichcan be achieved with the wild-type lac operon. The vector which yieldedthe lowest level of expression under repressed conditions was pLAC11,while the vector which yielded the highest level of expression underinduced conditions was pLAC33. TABLE 6 β-galactosidase levels obtainedin different expression vectors grown under either repressed or inducedconditions # of Miller Units Observed Repressed Induced Vector SourceConditions Conditions Fold Induction pLAC11 F' 19 11209 590X pLAC22Plasmid 152 13315  88X pLAC33 F' 322 23443  73X pKK223-3 F' 92 11037120X pKK233-2 F' 85 10371 122X pTrc99A Plasmid 261 21381  82X pET-21(+)Plasmid 2929 16803  6X pET-21(+)/pLysE Plasmid 4085 19558  5XpET-21(+)/pLysS Plasmid 1598 20268  13X

[0119] The average values obtained for the four clones that were testedfrom each vector are listed in the table. Standard deviation is notshown but was less than 5% in each case. Induction ratios are expressedas the ratio of enzymatic activity observed at fully induced conditionsversus fully repressed conditions. The plasmid pLysE yielded unexpectedresults; it was expected to cause lower amounts of lacZ to be expressedfrom pET-21(+) under repressed conditions and, instead, higher amountswere observed. As a result, both pLysE and pLysS were restriction mappedto make sure that they were correct.

[0120] Demonstrating that pLAC11 constructs can be tightly regulated.pLAC11 was designed to provide researchers with an expression vectorthat could be utilized to conduct physiological experiments in which acloned gene is studied under completely repressed conditions where it isoff or partially induced conditions where it is expressed atphysiologically relevant levels. FIG. 5 demonstrates how a pLAC11-lacZconstruct can be utilized to mimic chromosomally expressed lacZ thatoccurs under various physiological conditions by varying the amount ofIPTG inducer that is added. ALS226 cells containing pLAC11-lacZ weregrown to mid-log in rich media that contained varying amounts of IPTGand then β-galactosidase activity was assayed. Also indicated in thegraph are the average β-galactosidase activities obtained for strainswith a single chromosomal copy of the wild-type lacZ gene that weregrown under different conditions.

[0121] To demonstrate just how regulable pLAC11 is, the recA gene wascloned into the pLAC11 vector and transformed into cells which containeda null recA allele in the chromosome. As the results in Table 7 clearlyshow, recombination cannot occur in a host strain which contains anonfunctional RecA protein and thus P1 lysates which provide a Tn10dKantransposon cannot be used to transduce the strain to Kan^(R) at a highfrequency. recA⁻ cells which also contain the pLAC11-recA construct canbe transduced to Kan^(R) at a high frequency when grown under inducedconditions but cannot be transduced to Kan^(R) when grown underrepressed conditions. TABLE 7 The recombination (−) phenotype of a recAnull mutant strain can be preserved with a pLAC11-recA (wild-type)construct under repressed conditions Repressed Conditions InducedConditions Number of Kan^(R) Number of Kan^(R) Strain transductantstransductants ALS225 (recA⁺) 178,000 182,000 ALS514 (recA⁻)     5     4ALS515     4 174,000 (recA⁻ pLAC11-recA)

[0122] The data presented in Table 7 are the number of Kan^(R)transductants that were obtained from the different MC1061 derivativestrains when they were transduced with a P1 lysate prepared from strainALS598 which harbored a Tn10dKan transposon insertion. Overnights wereprepared from each of these strains using either rich medium to whichglucose was added at a final concentration of 0.2% (repressedconditions) or rich medium to which IPTG was added at a finalconcentration of 1 mM (induced conditions). The overnights were thendiluted 1 to 10 into the same medium which contained CaCl₂ added to afinal concentration of 10 mM and aerated for two hours to make themcompetent for transduction with P1 phage. Cells were thenspectrophotometrically normalized and aliquots of 5 OD₅₅₀ cellequivalents in a volume of approximately 0.1 ml were transduced with 0.1ml of concentrated P1 lysate as well as 0.1 ml of P1 lysates that hadbeen diluted to 10⁻¹, 10⁻², or 10⁻³. 0.2 ml of 0.1 M Sodium Citrate wasadded to the cell/phage mixtures and 0.2 ml of the final mixtures wereplated onto Rich Kanamycin plates and incubated overnight at 37° C. Thetotal number of Kan^(R) colonies were then counted. ALS225 recA⁺ datapoints were taken from the transductions which used the 10⁻³ dilutedphage, while ALS514 recA⁻ data points were taken from the transductionswhich used the concentrated phage. The data points for ALS515 recA⁻pCyt-3-recA grown under repressed conditions were taken from thetransductions which used the concentrated phage, while the data pointsfor ALS515 recA⁻ pCyt-3-recA grown under induced conditions were takenfrom the transductions which used the 10⁻³ diluted phage.

[0123] Testing various sources of LacI for trans repression of pLAC11.Because pLAC11 was designed to be used with an extraneous source of LacIrepressor, different episomal or plasmid sources of LacI which areroutinely employed by researchers were tested. Since one of the LacIsources also contained the lacZ gene, a reporter construct other thanpLAC11-lacZ was required and thus a pLAC11-xylE construct wasengineered. Table 8 shows the results of this study.

[0124] All of the LacI sources that were tested proved to be adequate torepress expression from pLAC11, however, some were better than others.The basal level of expression that was observed with F's which providedlacIq¹ or with the plasmid pMS421 which provided lacIq at approximatelysix copies per cell was lower than the basal level. of expression thatwas observed with F's which provided lacIq all three times that theassay was run. Unfortunately, however, the xylE gene could not beinduced as high when lacIq¹ on a F' or lacIq on a plasmid was used asthe source of Lac repressor. TABLE 8 Catechol 2,3-dioxygenase levelsobtained for a pLAC11-xylE construct when Lac repressor is provided byvarious sources Catechol 2,3-dioxygenase activity in milliunits/mgRepressed Induced Strain Source of LacI Conditions Conditions ALS224None 32.7 432.8 ALS535 F'lacIqΔ(lacZ)M15 .3 204.4 proA+B+ Tn10 ALS527F'lacIqΔ(lacZ)M15 .3 243.3 proA+B+ ALS227 pMS421 lacI^(q) .2 90.9 ALS225F'lacIq¹ Z⁺ Y⁺ A⁺ .2 107.4 ALS226 F'lacIq¹ Z::Tn5 Y⁺ .2 85.1 A⁺

[0125] The wild-type xylE gene was cloned into the pLAC11 vector and theresulting pLAC11-xylE construct was then transformed into each of theMC1061 derivative strains listed in the table. Rich overnights werediluted 1 to 200 in either Rich Glucose or Rich IPTG media and grownuntil they reached mid-log (OD₅₅₀=0.5). Cell extracts were then preparedand catechol 2,3-dioxygenase assays were performed as described byZukowski, et. al. (Proc. Natl. Acad. Sci. U. S. A. 80:1101-1105 (1983)).The average values obtained in three different experiments are listed inthe table. Standard deviation is not shown but was less 15 than 10% ineach case.

[0126] Discussion

[0127] Most of the routinely employed expression vectors rely on laccontrol in order to overproduce a gene of choice. The lacpromoter/operator functions as it does due to the interplay of threemain components. First, the wild-type lac-10 region (TATGTT) is veryweak. c-AMP activated CAP protein is able to bind to the CAP site justupstream of the −35 region which stimulates binding of RNA polymerase tothe weak −10 site. Repression of the lac promoter is observed whenglucose is the main carbon source because very little c-AMP is presentwhich results in low amounts of available c-AMP activated CAP protein.When poor carbon sources such as lactose or glycerol are used, c-AMPlevels rise and large amounts of c-AMP activated CAP protein becomeavailable. Thus induction of the lac promoter can occur. Second, Lacrepressor binds to the lac operator. Lac repressor can be overcome byallolactose which is a natural byproduct of lactose utilization in thecell, or by the gratuitous inducer, IPTG Third, the lac operator canform stable loop structures which prevents the initiation oftranscription due to the interaction of the Lac repressor with the lacoperator (O1) and one of two auxiliary operators, O2 which is locateddownstream in the coding region of the lacZ gene, or O3 which is locatedjust upstream of the CAP binding site.

[0128] While binding of Lac repressor to the lac operator is the majoreffector of lac regulation, the other two components are notdispensable. However, most of the routinely used lac regulable vectorseither contain mutations or deletions which alter the affect of theother two components. The pKK223-3 (J. Brosius et al., Proc. Natl. Acad.Sci. USA. 81:6929-6933 (1984)), pKK233-2 (E. Amann et al., Gene.40:183-190 (1985)), pTrc99A (E. Amann et al., Gene. 69:301-315 (1988)),and pET family of vectors (F. Studier, Method Enzymol. 185:60-89 (1990))contain only the lac operator (O1) and lack both the CAP binding site aswell as the O3 auxiliary operator. pKK223-3, pKK233-2, and pTrc99 use atrp-lac hybrid promoter that contains the trp-35 region and thelacUV5-10 region which contains a strong TATAAT site instead of the weakTATGTT site. The pET family of vectors use the strong T7 promoter. Giventhis information, perhaps it is not so surprising researchers have foundit is not possible to tightly shut off genes that are cloned into thesevectors.

[0129] The purpose of the studies described in Example I was to design avector which would allow researchers to better regulate their clonedgenes in order to conduct physiological experiments. The expressionvectors described herein were designed utilizing the wild-type lacpromoter/operator in order to accomplish this purpose and include all ofthe lac control region, without modification, that is contained betweenthe start of the O3 auxiliary operator through the end of the O1operator. As with all lac based vectors, the pLAC11, pLAC22, and pLAC33expression vectors can be turned on or off by the presence or absence ofthe gratuitous inducer IPTG.

[0130] Because the new vector, pLAC11, relies on the wild-type laccontrol region from the auxiliary lac O3 operator through the lac O1operator, it can be more tightly regulated than the other availableexpression vectors. In direct comparison studies with pKK223-3,pKK233-2, pTrc99A, and pET-21(+), the lowest level of expression underrepressed conditions was achievable with the pLAC11 expression vector.Under fully induced conditions, pLAC11 expressed lacZ protein that wascomparable to the levels achievable with the other expression vectors.Induction ratios of 1000× have been observed with the wild-type lacoperon. Of all the expression vectors that were tested, only pLAC11yielded induction ratios which were comparable to what has been observedwith the wild-type lac operon. It should be noted that the regulationachievable by pLAC11 may be even better than the data in Table 6indicates. Because lacZ was used in this test, the auxiliary lac O2operator which resides in the coding region of the lacZ gene wasprovided to the pKK223-3, pKK233-2, pTrc99A, and pET-21(+) vectors whichdo not normally contain either the O2 or O3 auxiliary operators. Thusthe repressed states that were observed in the study in Table 6 areprobably lower than one would normally observe with the pKK223-3,pKK233-2, pTrc99A, and pET-21(+) vectors.

[0131] To meet the expression needs required under differentexperimental circumstances, two additional expression vectors which arederivatives of pLAC11 were designed. pLAC22 provides lacIq on the vectorand thus unlike pLAC11 does not require an extraneous source of LacI forits repression. pLAC33 contains the mutated ColEl replicon from pUC8 andthus allows proteins to be expressed at much higher levels due to theincrease in the copy number of the vector. Of all the expressions thatwere evaluated in direct comparison studies, the highest level ofprotein expression under fully induced conditions was achieved using thepLAC33 vector. Because the cloning regions are identical in pLAC11,pLAC22, and pLAC33, genes that are cloned into one of these vectors canbe trivially subcloned into either of the other two vectors depending onexperimental circumstances. For physiological studies, pLAC11 is thebest suited of the three vectors. If, however, the bacterial strain ofchoice can not be modified to introduce elevated levels of Lac repressorprotein which can be achieved by F's or compatible plasmids that providelacIq or lacIq¹, the pLAC22 vector can be utilized. If maximaloverexpression of a gene product is the goal, then the pLAC33 vector canbe utilized.

[0132] Numerous experiments call for expression of a cloned gene productat physiological levels; i.e., at expression levels that are equivalentto the expression levels observed for the chromosomal copy of the gene.While this is not easily achievable with any of the commonly utilizedexpression vectors, these kinds of experiments can be done with thepLAC11 expression vector. By varying the IPTG concentrations, expressionfrom the pLAC11 vector can be adjusted to match the expression levelsthat occur under different physiological conditions for the chromosomalcopy of the gene. In fact, strains which contain both a chromosomal nullmutation of the gene in question and a pLAC11 construct of the genepreserve the physiological phenotype of the null mutation underrepressed conditions.

[0133] Because the use of Lac repressor is an essential component of anyexpression vector that utilizes the lac operon for its regulation, theability of different source of LacI to repress the pLAC11 vector wasalso investigated. Researchers have historically utilized either lacIqconstructs which make 10 fold more Lac repressor than wild-type lacI orlacIq¹ constructs which make 100 fold more Lac repressor than wild-typelacI (B. Müller-Hill, Prog. Biophys. Mol. Biol. 30:227-252 (1975)). Thegreatest level of repression of pLAC11 constructs could be achievedusing F's which provided approximately one copy of the lacIq¹ gene or amulticopy compatible plasmid which provided approximately six copies ofthe lacIq gene. However, the induction that was achievable using theselacI sources was significantly lower than what could be achieved whenF's which provided approximately one copy of the lacIq¹ gene were usedto repress the pLAC11 construct. Thus if physiological studies are thegoal of an investigation, then F's which provide approximately one copyof the lacIq¹ gene or a multicopy compatible plasmid which providesapproximately six copies of the lacIq gene can be used to regulate thepLAC11 vector. However, if maximal expression is desired, then F's whichprovide approximately one copy of the lacIq gene can be utilized.Alternatively, if a bacterial strain can tolerate prolongedoverexpression of an expressed gene, and overexpression of a geneproduct is the desired goal, then maximal expression under inducedconditions is obtained when a bacteria strain lacks any source of Lacrepressor.

Example II An in Vivo Approach for Generating Novel Bioactive Peptidesthat Inhibit the Growth of E. coli

[0134] A randomized oligonucleotide library containing sequences capableof encoding peptides containing up to 20 amino acids was cloned intopLAC11 (Example I) which allowed the peptides to either be tightlyturned off or overproduced in the cytoplasm of E. coli. The randomizedlibrary was prepared using a [NNN] codon design instead of either the[NN(G, T)] or [NN(G, C)] codon design used by most fusion-phagetechnology researchers. [NN(G, T)] or [NN(G, C)] codons have been widelyused instead of [NNN] codons to eliminate two out of the three stopcodons, thus increasing the amount of full-length peptides that can besynthesized without a stop codon (J. Scott et al., Science 249:386-390(1990); J. Delvin et al., Science 249:404-406 (1990); S. Cwirla et al.,Proc. Nat'l. Acad. Sci. U. S. A. 87:6378-6382 (1990)). However, the[NN(G, T)] and [NN(G, C)] oligonucleotide codon schemes eliminate halfof the otherwise available codons and, as a direct result, biases thedistribution of amino acids that are generated. Moreover, the [NN(G, T)]and [NN(G, C)] codon schemes drastically affect the preferential codonusage of highly expressed genes and removes a number of the codons whichare utilized by the abundant tRNAs that are present in E. coli (H.Grosjean et al., Gene. 18: 199-209 (1982); T. Ikemura, J. Mol. Biol.151:389-409 (1981)).

[0135] Of the 20,000 peptides screened in this Example, 21 inhibitors ofcell growth were found which could prevent the growth of E. coli onminimal media. The top twenty inhibitor peptides were evaluated forstrength of inhibition, and the putative amino acid sequences of the top10 “anchorless” inhibitor peptides were examined for commonly sharedfeatures or motifs.

[0136] Materials and Methods

[0137] Media. Rich LB and minimal M9 media used in this study wasprepared as in Example I. Ampicillin was used in rich media at a finalconcentration of 100 îg/ml and in minimal media at a final concentrationof 50 îg/ml. WPTG was added to media at a final concentration of 1 mM.

[0138] Chemicals and Reagents. Extension reactions were carried outusing Klenow from New England Biolabs while ligation reactions wereperformed using T4 DNA Ligase from Life Sciences. IPTG was obtained fromDiagnostic Chemicals Limited.

[0139] Bacterial Strains and Plasmids. ALS225, which isMC1061/F'lacIq¹Z+Y+A+ (see Example I), was the E. coli bacterial strainused in this Example. The genotype for MC1061 is araD139Δ(araABOIC-leu)7679Δ(lac)X74 galU galK rpsL hsr−hsm+ (M. Casadaban etal., J. Mol. Biol. 138: 179-207 (1980)). pLAC11, a highly regulableexpression vector, is described in Example I.

[0140] Generation of the Randomized Peptide Library. The 93 baseoligonucleotide 5′TAC TAT AGA TCT ATG (NNN)₂₀ TAA TAA GAA TTC TCG ACA 3′(SEQ ID NO:23), where N denotes an equimolar mixture of the nucleotidesA, C, G, or T, was synthesized with the trityl group and subsequentlypurified with an OPC cartridge using standard procedures. Thecomplementary strand of the 93 base oligonucleotide was generated by anextension/fill-in reaction with Klenow using an equimolar amount of the18 base oligonucleotide primer 5′TGT CGA GAA TTC TTA TTA 3′ (SEQ IDNO:24). After extension, the resulting ds-DNA was purified using aPromega DNA clean-up kit and restricted with EcoR I and Bgl II (Promega,Madison, Wis.). The digested DNA was again purified using a Promega DNAclean-up kit and ligated to pLAC11 vector which had been digested withthe same two restriction enzymes. The resulting library was transformedinto electrocompetent ALS225 E. coli cells under repressed conditions(LB, ampicillin, plus glucose added to 0.2%).

[0141] Screening of Transformants to Identify Inhibitor Clones.Transformants were screened to identify any that could not grow onminimal media when the peptides were overproduced. Using this scheme,any transformant bacterial colony that overproduces a peptide thatinhibits the production or function of a protein necessary for growth ofthat transformant on minimal media will be identified. Screening onminimal media, which imposes more stringent growth demands on the cell,will facilitate the isolation of potential inhibitors from the library.It is well known that growth in minimal media puts more demands on abacterial cell than growth in rich media as evidenced by the drasticallyreduced growth rate; thus a peptide that adversely affects cell growthis more likely to be detected on minimal media. Screening was carriedout using a grid-patching technique. Fifty clones at a time were patchedonto both a rich repressing plate (LB Amp glucose) and a minimalinducing plate (M9 glycerol Amp IPTG) using an ordered grid. Patchesthat do not grow are sought because presumably these represent bacteriathat are being inhibited by the expressed bioactive peptide. To verifythat all of the inhibitors were legitimate, plasmid DNA was made fromeach inhibitory clone (QIA Prep Spin Miniprep kit; Qiagen Cat. No.27104) and transformed into a fresh background (ALS225 cells), thenchecked to confirm that they were still inhibitory on plates and thattheir inhibition was dependent on the presence of the inducer, IPTG.

[0142] Growth Rate Analysis in Liquid Media. Inhibition strength of thepeptides was assessed by subjecting the inhibitory clones to a growthrate analysis in liquid media. To determine the growth rate inhibition,starting cultures of both the peptides to be tested and a control strainwhich contains pLAC11 were diluted from a saturated overnight culture toan initial OD₅₅₀ of ˜0.01. All cultures were then induced with 1 mM IPTGand OD₅₅₀ readings were taken until the control culture reached an OD₅₅₀of ˜0.5. The hypothetical data in Table 9 shows that when the controlstrain reaches an OD₅₅₀ of about 0.64 (at about 15 hours), a strainwhich contains a peptide that inhibits the growth rate at 50% will onlyhave reached an OD₅₅₀ of only about 0.08. Thus, the growth of a 50%inhibited culture at 15 hours (i.e., the OD₅₅₀ at 15 hours, which isproportional to the number of cells in a given volume of culture) isonly about 12.5% (that is, 0.08/0.64×100) of that of a control strainafter the same amount of time, and the inhibitor peptide would thus haveeffectively inhibited the growth of the culture (as measured by theOD₅₅₀ at the endpoint) by 87.5% (=100%−12.5%). TABLE 9 Hypothetical datafrom a peptide that inhibits growth rate at 30%, 50% and 70% OD550readings on 0D550 readings on a a culture which contains a peptide Timein control culture which that inhibits the growth rate at . . . Hourscontains pLAC11 25% 50% 75% 0 .010 .010 .010 .010 2.5 .020 .017 .015.012 5 .040 .028 .020 .014 7.5 .080 .047 .030 .017 10 .160 .079 .040.020 12.5 .320 .133 .060 .024 15 .640 .226 .080 .028

[0143] An example is shown in FIG. 6, wherein ALS225 cells containingthe pLAC11 vector (control), and either the one day inhibitor pPep1 orthe two day inhibitor pPep12 (see below), were grown in minimal M9glycerol media with IPTG added to 1 mM. OD₅₅₀ readings were then takenhourly until the cultures had passed log phase. Growth rates weredetermined by measuring the spectrophotometric change in OD₅₅₀ per unittime within the log phase of growth. The inhibition of the growth ratewas then calculated for the inhibitors using pLAC11 as a control.

[0144] Sequencing the Coding Regions of the Inhibitor Peptide Clones.The forward primer 5′TCA TTA ATG CAG CTG GCA CG 3′ (SEQ ID NO:25) andthe reverse primer 5′TTC ATA CAC GGT GCC TGA CT 3′ (SEQ ID NO:26) wereused to sequence both strands of the top ten “anchorless” inhibitorpeptide clones identified by the grid-patching technique. If anerror-free consensus sequence could not be deduced from these twosequencing runs, both strands of the inhibitor peptide clones inquestion were resequenced using the forward primer 5′TAG CTC ACT CAT TAGGCA CC 3′ (SEQ ID NO:27)and the reverse primer 5′GAT GAC GAT GAG CGC ATTGT 3′ (SEQ ID NO:28). The second set of primers were designed to annealdownstream of the first set of primers in the pLAC11 vector.

[0145] Generating Antisense Derivatives of the Top Five “Anchorless”Inhibitor Clones. Oligonucleotides were synthesized which duplicated theDNA insert contained between the Bgl II and EcoR I restriction sites forthe top five “anchorless” inhibitor peptides as shown in Table 12 withone major nucleotide change. The “T” of the ATG start codon was changedto a “C” which resulted in an ACG which can not be used as a startcodon. The oligonucleotides were extended using the same 18 baseoligonucleotide primer that was used to build the original peptidelibrary. The resulting ds-DNA was then restricted, and cloned intopLAC11 exactly as described in the preceding section “Generating therandomized peptide library.” The antisense oligonucleotides that wereused are as follows: pPepI(antisense): 5′TAC TAT AGA TCT ACG GTC ACT GAATTT TGT GGC TTG (SEQ ID NO: 29) TTG GAC CAA CTG CCT TAG TAA TAG TGG AAGGCT GAA ATT AAT AAG AAT TCT CGA CA 3′; pPepS(antisense): 5′TAC TAT AGATCT ACG TGG CGG GAC TCA TGG ATT AAG (SEQ ID NO: 30) GGT AGG GAC GTG GGGTTT ATG GGT TAA AAT AGT TTG ATA ATA AGA ATT CTC GAC A 3′pPep12(antisense): 5′TAC TAT AGA TCT ACG AAC GGC CGA ACC AAA CGA ATC(SEQ ID NO: 31) CGG GAC CCA CCA GCC GCC TAA ACA GCT ACC AGC TGT GGT AATAAG AAT TCT CGA CA 3′ pPep13(antisense): 5′TAC TAT AGA TCT ACG GAC CGTGAA GTG ATG TGT GCG (SEQ ID NO: 32) GCA AAA CAG GAA TGG AAG GAA CGA ACGCCA TAG GCC GCG TAA TAA GAA TTC TCG ACA 3′ pPep19(antisense): 5′TAC TATAGA TCT ACG AGG GGC GCC AAC TAA GGG GGG (SEQ ID NO: 33) GGG AAG GTA TTTGTC CCG TGC ATA ATC TCG GGT GTT GTC TAA TAA GAA TTC TCG ACA 3′

[0146] Results

[0147] Identifying and Characterizing Inhibitor Peptides from theLibrary. Approximately 20,000 potential candidates were screened asdescribed hereinabove, and 21 IPTG-dependent growth inhibitors wereisolated. All the inhibitors so identified were able to prevent thegrowth of the E. coli bacteria at 24 hours, and three of the 21inhibitors were able to prevent the growth of the E. coli bacteria at 48hours, using the grid patching technique. These three inhibitors wereclassified as “two day” inhibitors; the other 18 were classified as“one-day” inhibitors.

[0148] Results from the growth rate analysis for candidate peptideinhibitors are shown in Table 10. The % inhibition of the growth ratewas calculated by comparing the growth rates of cells that containedinduced peptides with the growth rate of cells that contained theinduced pLAC11 vector. Averaged values of three independentdeterminations are shown. TABLE 10 Ability of the Inhibitor Peptides toInhibit Cell Growth % Inhibitor Type Inhibition pLAC11 — 0 (control)pPep1 1 Day 25 pPep2 1 Day 23 pPep3 2 Day 80 pPep4 1 Day 21 pPep5 1 Day24 pPep6 1 Day 27 pPep7 1 Day 26 pPep8 1 Day 29 pPep9 1 Day 22 pPep10 1Day 24 pPep11 1 Day 22 pPep12 2 Day 82 pPep13 1 Day 28 pPep14 2 Day 71pPep15 1 Day 23 pPep16 1 Day 24 pPep17 1 Day 28 pPep18 1 Day 24 pPep19 1Day 29 pPep20 1 Day 19 pPep21 1 Day 23

[0149] Of the 21 peptides that were tested, the one-day inhibitorpeptides inhibited the bacterial growth rate at a level of approximately25%, while the two-day inhibitor peptides inhibited the bacterial growthrate at levels greater than 75%. As can be seen from the hypotheticaldata in Table 9, a one-day inhibitor which inhibited the growth rate at25% would have only reached an OD₅₅₀ of 0.226 when the control strainreached an OD₅₅₀ of 0.64. At that point in time, the growth of theculture that is inhibited by a one-day inhibitor (as measured by theend-point OD₅₅₀) only be only 35.3% of that of a control strain at thatpoint; thus the inhibitor peptide would have effectively inhibited thegrowth of the culture by 64.7%. A two-day inhibitor which inhibited thegrowth rate at 75% would have only reached an OD₅₅₀ of 0.028 when thecontrol strain reached an OD₅₅₀ of 0.64. Thus the growth of the culturethat is being inhibited by a two-day inhibitor will only be 4.4% of thatof the control strain at this point, and the inhibitor peptide wouldhave effectively inhibited the growth of the culture by 95.6%. Thesecalculations are consistent with the observation that two-day inhibitorsprevent the growth of bacteria on plates for a full 48 hours while theone-day inhibitors only prevent the growth of bacteria on plates for 24hours.

[0150] All 21 candidates were examined using restriction analysis todetermine whether they contained 66 bp inserts as expected. While mostof them did, the two-day inhibitors pPep3 and pPep14 were found tocontain a huge deletion. Sequence analysis of these clones revealed thatthe deletion had caused the carboxy-terminal end of the inhibitorpeptides to become fused to the amino-terminal end of the short 63 aminoacid Rop protein. The rop gene, which is part of the ColEl replicon, islocated downstream from where the oligonucleotide library is insertedinto the pLAC11 vector.

[0151] Sequence Analysis of the Top 10 “Anchorless” Inhibitor Peptides.The DNA fragments comprising the sequences encoding the top 10“anchorless” inhibitor peptides (i.e., excluding the two Rop fusionpeptides) were sequenced, and their coding regions are shown in Table11. Stop codons are represented by stars, and the landmark Bgl II andEcoR I restriction sites for the insert region are underlined. Since theends of the oligonucleotide from which these inhibitors were constructedcontained these restriction sites, the oligonucleotide was not gelisolated when the libraries were prepared in order to maximize theoligonucleotide yields. Because of this, several of the inhibitoryclones were found to contain one (n−1) or two (n−2) base deletions inthe randomized portion of the oligonucleotide. TABLE 11 Sequenceanalysis of the insert region from the top 10 inhibitory clones > andthe peptides that they are predicted to encode pPep1-13 aa GAG GAAAGA TCT ATG GTC ACT GAA TTT TGT GGC TTG TTG GAG CAA CTG CCT TAG TAA TAGTGG AAG (SEQ ID NO: 35)                M   V   T   E   F   C   G   L   L   D   Q   L   P   *   *   *(SEQ ID NO: 34) GCT GAA ATT AAT AAG AAT TC pPep5-16 aa CAG GAAAGA TCT ATG TGG CGG GAC TCA TGG ATT AAG GGT AGG GAC GTG GGG TTT ATG GGTTAA AAT (SEQ ID NO: 37)                M   W   R   D   S   W   I   K   G   R   D   V   G   F   N   G   *(SEQ ID NO: 36) AGT TTG ATA ATA AGA ATT C pPep6-42 aa-last 25 aa couldform a hydrophobic membrane-spanning domain CAG GAA AGA TCT ATG TCA GGGGGA CAT GTG ACG AGG GAG TGG AAG TCG GCG ATG TCC AAT CGT TGG (SEQ ID NO:39)                M   S   G   G   N   V   T   K   E   C   K   S   A   N   S   N   R   W(SEQ ID NO: 38) ATC TAC GTA ATA AGA ATT CTC ATG TTT GAG ACC TTA TGA TGGATA AGC TTT AAT GCG GTA GTT TATI   Y   V   I   K   I   L   N   F   D   S   L   S   S   I   S   F   N   A   V   V   YCAC AGT TAA H   S   * pPep7-6 aa CAG GAA AGA TCT ATG TAT TTG TTC ATC GGATAA TAC TTA ATG GTC CGC TGG AGA ACT TGA GTT TAA (SEQ ID NO: 41)               M   Y   L   F   I   G   * (SEQ ID NO: 40) TAA GAA TTCpPep8-21 aa CAG GAA AGA TCT ATG CTT CTA TTT GGG GGG GAC TGC GGG GAG AAAGGG GGA TAG TTT ACT GTG CTA (SEQ ID NO: 42)                M   L   L   F   G   G   D   C   G   Q   K   A   G   Y   F   T   V   L(SEQ ID NO: 43) CCG TGA AGG TAA TAA GAA TTC  P   S   R   *   * pPep10-20aa-predicted to be 45% β-sheet-amino acids 6-14 CAG GAA AGA TCT ATG ATTGGG GGA TCG TTG AGG TTG CCC TGG GCA ATA GTT TGT AAT AAG AAT TCT (SEQ IDNO: 44)                M   I   G   G   S   L   S   F   A   W   A   I   V   C   N   K   N   S(SEQ ID NO: 45) CAT GTT TGA N   V   * pPep12-14 aa CAG GAA AGA TCT ATGAAC GGC CGA ACC AAA CGA ATC CGG GAC CCA CCA GCC GCC TAA ACA GCT ACC (SEQID NO: 47)                M   N   G   H   T   K   R   I   R   D   P   P   A   A   *(SEQ ID NO: 46) AGC TGT GGT AAT AAG AAT TC  pPep13-18 aa-predicted to be72% α-helical-amino acids 3-15 CAG GAA AGA TCT ATG GAC CGT GAA GTG ATGTGT GCG GCA AAA CAG GAA TGG AAG GAA CGA ACG CCA (SEQ ID NO: 49)                M   D   R   E   V   N   C   A   A   K   Q   E   W   K   E   R   T   P(SEQ ID NO: 48) TAG GCC GCG TAA TAA GAA TTC  * pPep17-12 aa CAG GAAAGA TCT ATG TAG CCC AAT GCA CTG GGA GCA CGC GTG TTA GGT CTA GAA GCC ACGTAC CCA (SEQ ID NO: 50)                M   *                               M   L   G   L   E   A   T   Y   P(SEQ ID NO: 51) TTT AAT CCA TAA TAA GAA TTC  F   N   P   *   * pPep19-5aa CAG GAA AGA TCT ATG AGG GGC CCC AAC TAA GGG GGG GGG AAG GTA TTT GTCCCG TGC ATA ATC TCG (SEQ ID NO: 53)                M   R   G   A   N   * (SEQ ID NO: 52) GGT GTT GTC TAATAA GAA TTC 

[0152] Eight out of the top 10 inhibitors were predicted to encodepeptides that terminate before the double TAA TAA termination site,which was engineered into the oligonucleotide. Two of the inhibitors,pPep6 and pPep10, which contain deletions within the randomized portionof the oligonucleotide, are terminated beyond the EcoR I site. One ofthe inhibitors, pPep17, contains a termination signal just after the ATGstart codon. However, just downstream from this is a Shine Dalgamo siteand a GTG codon, which should function as the start codon.Interestingly, the start sites of several proteins such as Rop areidentical to that proposed for the pPep17 peptide (G. Cesareni et al.,Proc. Natl. 35 Acad. Sci. USA. 79:6313-6317 (1982)). The average andmedian length for the 8 peptides whose termination signals occurredbefore or at the double TAA TAA termination site was 13 amino acids.

[0153] The characteristics of the predicted coding regions of theinhibitor peptides proved to be quite interesting. Three out of the top10 peptides, pPep1, pPep13, and pPep17, contained a proline residue astheir last (C-terminal) amino acid. Additionally, one of the peptides,pPep12, contained 2 proline residues near the C-terminus, at the n−2 andn−3 positions. Thus there appears to be a bias for the placement ofproline residues at or near the end of several of the inhibitorypeptides. Secondary structure analysis predicted that 3 out of the 10peptides contained a known motif that could potentially form a verystable structure. pPep13, a peptide containing a C-terminal proline, ispredicted to be 72% α-helical, pPep10 is predicted to be 45% β-sheet,and pPep6 is predicted to form a hydrophobic membrane spanning domain.

[0154] Verifying that the Inhibitory Clones do not Function asAntisense. To rule out the possibility that the bioactivity of theinhibitory clones resulted from their functioning as antisense RNA orDNA (thus hybridizing to host DNA or RNA) rather than by way of theencoded peptides, the insert regions between the Bgl II and EcoR I sitesfor the top five inhibitors from Table 10 were recloned into the pLAC11vector using oligonucleotides which converted the ATG start codon to anACG codon thus abolishing the start site. In all five cases the newconstructs were no longer inhibitory (see Table 12), thus confirmingthat it is the encoded peptides that causes the inhibition and not theDNA or transcribed mRNA. TABLE 12 Antisense test of the top 5“anchorless” inhibitory peptides % inhibition % inhibition versus pLAC11Antisense versus pLAC11 Inhibitory peptide control construct controlpPep1 26 pPep1-anti 0 pPep5 23 pPep5-anti 0 pPep12 80 pPep12-anti 0pPep13 28 pPep13-anti 0 pPep19 29 pPep19-anti 0

[0155] Growth rates for cells containing the induced inhibitors orantisense constructs were determined and then the % inhibition wascalculated by comparing these values to the growth rate of cells thatcontained the induced pCyt-3 vector.

[0156] Discussion

[0157] Use of the tightly regulable pLAC11 expression vector madepossible the identification of novel bioactive peptides. The bioactivepeptides identified using the system described in this Example inhibitthe growth of the host organism (E. coli) on minimal media. Moreover,bioactive peptides thus identified are, by reason of the selectionprocess itself, stable in the host's cellular environment. Peptides thatare unstable in the host cell, whether or not bioactive, will bedegraded; those that have short half-lives are, as a result, not part ofthe selectable pool. The selection system thus makes it possible toidentify and characterize novel, stable, degradation-resistant bioactivepeptides in essentially a single experiment.

[0158] The stability of the inhibitory peptides identified in thisExample may be related to the presence of certain shared structuralfeatures. For example, three out of the top 10 inhibitory “anchorless”(i.e., non-Rop fusion) peptides contained a proline residue as theirlast amino acid. According to the genetic code, a randomly generatedoligonucleotide such as the one used in this Example has only a 6%chance of encoding a proline at a given position, yet the frequency of aC-terminal proline among the top ten inhibitory peptides is a full 30%.This 5-fold bias in favor of a C-terminal proline is quite surprising,because although the presence of proline in a polypeptide chaingenerally protects biologically active proteins against nonspecificenzymatic degradation, a group of enzymes exists that specificallyrecognize proline at or near the N- and C-termini of peptide substrates.Indeed, proline-specific peptidases have been discovered that coverpractically all situations where a proline residue might occur in apotential substrate (D. F. Cunningham et al., Biochimica et BiophysicsActa 1343:160-1 86 (1997)). For example, although the N-terminalsequences Xaa-Pro-Yaa- and Xaa-Pro-Pro-Yaa (SEQ ID NO:54) have beenidentified as being protective against nonspecific N-terminaldegradation, the former sequence is cleaved by aminopeptidase P (at theXaa-Pro bond) and dipeptidyl peptidases IV and II (at the-Pro-Yaa-bond)) (Table 5, G. Vanhoof et al., FASEB J. 9:736-44 (1995);D. F. Cunningham et al., Biochimica et Biophysics Acta 1343:160-186(1997)); and the latter sequence, present in bradykinin, interleukin 6,factor XII and erythropoietin, is possibly cleaved by consecutive actionof aminopeptidase P and dipeptidyl peptidase IV (DPPIV), or by prolyloligopeptidase (post Pro-Pro bond) (Table 5, G. Vanhoof et al., FASEB J.9:736-44 (1995)). Prolyl oligopeptidase is also known to cleave Pro-Xaabonds in peptides that contain an N-terminal acyl-Yaa-Pro-Xaa sequence(D. F. Cunningham et al., Biochimica et Biophysics Acta 1343:160-186(1997)). Other proline specific peptidases acting on the N-terminus ofsubstrates include prolidase, proline iminopeptidase and prolinase.Prolyl carboxypeptidase and carboxypeptidase P, on the other hand,cleave C-terminal residues from peptides with proline being thepreferred P₁ residue (D. F. Cunningham et al., Biochimica et BiophysicsActa 1343:160-186 (1997).

[0159] Also of interest with respect to the stability of the inhibitorypeptides, three of the top ten (30%) contained motifs that werepredicted, using standard protein structure prediction algorithms, toform stable secondary structures. One of the peptides (which also has aC-terminal proline) was predicted to be 72% α-helical. Another waspredicted to be 45% β-sheet; this peptide may dimerize in order toeffect the hydrogen bonding necessary to form the β-sheet. A third waspredicted to possess a hydrophobic membrane spanning domain. Accordingto these algorithms (see, e.g., P. Chou et al., Adv. Enzymol. 47:45-148(1978); J. Gamier et al., J. Mol. Biol. 120:97-120 (1978); P. Chou,“Prediction of protein structural classes from amino acid composition,”In Prediction of Protein Structure and the Principles of ProteinConformation (Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586(1990); P. Klein et al., Biochim. Biophys. Acta 815:468-476 (1985)), arandomly generated oligonucleotide such as the one used in our studieswould have had no better than a 1 in a 1000 chance of generating themotifs that occurred in these peptides.

[0160] Finally, two of the three two-day inhibitors proved to be fusionpeptides in which the carboxyl terminus of the peptides was fused to theamino terminus of the Rop protein. Rop is a small 63 amino acid proteinthat consists of two antiparallel É-helices connected by a sharp hairpinloop. It is a dispensable part of the ColEl replicon which is used byplasmids such as pBr322, and it can be deleted without causing anyill-effects on the replication, partitioning, or copy numbers ofplasmids that contain a ColEl ori (X. Soberon, Gene. 9:287-305 (1980).Rop is known to possess a highly stable structure (W. Eberle et al.,Biochem. 29:7402-7407 (1990); S. Betz et al., Biochemistry 36:2450-2458(1997)), and thus it could be serving as a stable protein anchor forthese two peptides.

[0161] Table 13 lists naturally occurring bioactive peptides whosestructures have been determined. Most of these peptides contain orderedstructures, further highlighting the importance of structuralstabilization. Research on developing novel synthetic inhibitorypeptides for use as potential therapeutic agents over the last few yearshas shown that peptide stability is a major problem that must be solvedif designer synthetic peptides are to become a mainstay in thepharmaceutical industry (J. Bai et al., Crit. Rev. Ther. Drug.12:339-371 (1995); R. Egleton Peptides. 18:1431-1439 (1997); L. Wearley,Crit Rev Ther Drug Carrier Syst. 8:331-394 (1991). The system describedin this Example represents a major advance in the art of peptide drugdevelopment by biasing the selection process in favor of bioactivepeptides that exhibit a high degree of stability in an intracellularenvironment. TABLE 13 Structural motifs observed in naturally occurringbioactive peptides Bioactive Size in Structural Peptide Amino acidsMotif Reference Dermaseptin 34 α-helix Mor et al., Biochemistry, 33:6642-6650 (1994) Endorphin 30 α-helix Blanc et al., J. Biol. Chem., 258:8277-8284 (1983) Glucagons 29 α-helix Bedarkar et al., Ciba Found Symp60: 105-121 (1977) Magainins^(a) 23 α-helix Bechinger et al., ProteinSci. 2: 2077-2084 (1993) Mastoparan 14 α-helix Cachia et al.,Biochemistry 25: 3553-3562 (1986) Melittin 26 α-helix Terwilliger etal., J. Biol. Chem. 257: 6010-6015 (1982) Motilin 22 α-helix Khan etal., Biochemistry 29: 5743-5751 (1990) PK1 (5-24) 20 α-helix Reed etal., Biochemistry 26: 7641-7647 (1987) Secretin 27 α-helix Gronenborn,et al. FEBS Lett., 215: 88-94 (1987) Atrial Natriuretic 28 disulfidebonds Misono, et al., Biochem. Biophys. Peptide Res. Comm. 119: 524-529(1984) Calcitonin 32 disulfide bonds Barling et al., Anal. Biochem. 144:542-552 (1985) Conotoxins^(a) 10-30 disulfide bonds Olivera, et al., J.Biol. Chem. 266: 22067-22070 (1991) Defensins^(a) 29-34 disulfide bondsLehrer, et al., Ann. Intern. Med. 109: 127-142 (1988) EETI II 29disulfide bonds Heitz, et al., Biochemistry 28: 2392-2398 (1989)Oxytocin  9 disulfide bonds Urry, et al., Proc. Natl. Acad. Sci. USA 60:967-974 (1968) Somatostatin 14 disulfide bonds Namboodiri, et al. J.Biol. Chem. 257: 10030-10032 (1982) Vasopressin  9 disulfide bonds Fong,et al., Biochem. Biophys. Res. Comm. 14: 302-306 (1964) Bombesin 14disordered Carmona, et al., Biochim. Biophys. Acta 1246: 128-134 (1995)Histatin 24 disordered Xu, et al. J. Dent. Res. 69: 1717-1723 (1990)Substance P 11 disordered Williams and Weaver, J. Biol. Chem. 265:2505-2513 (1990)

Example III Directed Synthesis of Stable Synthetically EngineeredInhibitor Peptides

[0162] These experiments were directed toward increasing the number ofbioactive peptides produced by the selection method described in ExampleII. In, the initial experiment, randomized peptides fused to the Ropprotein, at either the N- or C-terminus, were evaluated. In the secondexperiment, nucleic acid sequences encoding peptides containing arandomized internal amino acid sequence flanked by terminal prolineswere evaluated. Other experiments included engineering into the peptidesan α-helical structural motif, and engineering in a cluster of oppositecharges at the N- and C-termini of the peptide.

[0163] Materials and Methods

[0164] Media. Rich LB and minimal M9 media used in this study wasprepared as described by Miller (see Example I). Ampicillin was used inrich media at a final concentration of 100 îg/ml and in minimal media ata final concentration of 50 îg/ml. IPTG was added to media at a finalconcentration of 1 mM.

[0165] Chemicals and Reagents. Extension reactions were carried outusing Klenow from New England Biolabs (Bedford, Mass.) while ligationreactions were performed using T4 DNA ligase from Life Sciences(Gaithersburg, Md.) Alkaline phosphatase (calf intestinal mucosa) fromPharmacia (Piscataway, N.J.) was used for dephosphorylation. IPTG wasobtained from Diagnostic Chemicals Limited (Oxford, Conn.).

[0166] Bacterial Strains and Plasmids. ALS225, which isMC1061/F'lacIq¹Z+Y+A+, was the E. coli bacterial strain used in thisstudy (see Example I). The genotype for MC1061 is araD139Δ(araABOIC-leu)7679 Δ(lac)X74 galU galK rpsL hsr−hsm+ as previouslydescribed. pLAC11 (Example I), a highly regulable expression vector, wasused to make p-Rop(C) and p(N)Rop-fusion vectors as well as the otherrandomized peptide libraries which are described below.

[0167] Construction of the p-Rop(C) Fusion Vector. The forward primer5′TAC TAT AGA TCT ATG ACC AAA CAG GAA AAA ACC GCC 3′ (SEQ ID NO:55) andthe reverse primer 5′TAT ACG TAT TCA GTT GCT CAC ATG TTC TTT CCT GCG 3′(SEQ ID NO:56)were used to PCR amplify a 558 bp DNA fragment usingpBR322 as a template. This fragment contained a Bgl II restriction sitewhich was incorporated into the forward primer followed by an ATG startcodon and the Rop coding region. The fragment extended beyond the Ropstop codon through the Afl III restriction site in pBR322. The amplifieddsDNA was gel isolated, restricted with Bgl II and Afl III, and thenligated into the pLAC expression vector which had been digested with thesame two restriction enzymes. The resulting p-Rop(C) fusion vector is2623 bp in size (FIG. 7).

[0168] Construction of the p(N)Rop-Fusion Vector. The forward primer5′AAT TCA TAC TAT AGA TCT ATG ACC AAA CAG GAA AAA ACC GC 3′ (SEQ IDNO:57) and the reverse primer 5′TAT ATA ATA CAT GTC AGA ATT CGA GGT TTTCAC CGT CAT CAC 3′ (SEQ ID NO:58) were used to PCR amplify a 201 bp DNAfragment using pBR322 as a template. This fragment contained a Bgl IIrestriction site which was incorporated into the forward primer followedby an ATG start codon and the Rop coding region. The reverse primerplaced an EcoR I restriction site just before the Rop TGA stop codon andan Afl III restriction site immediately after the Rop TGA stop codon.The amplified dsDNA was gel isolated, restricted with Bgl II and AflIII, and then ligated into the pLAC11 expression vector which had beendigested with the same two restriction enzymes. The resultingp(N)Rop-fusion vector is 2262 bp in size (FIG. 8).

[0169] Generation of Rop Fusion Randomized Peptide Libraries. Peptidelibraries were constructed as described in Example II. The syntheticoligonucleotide 5′TAC TAT AGA TCT ATG (NNN)₂₀ CAT AGA TCT GCG TGC TGTGAT 3′ (SEQ ID NO:59) was used to construct the randomized peptidelibraries for use with the p-Rop(C) fusion vector, substantially asdescribed in Example II. The complementary strand of thisoligonucleotide was generated by a fill-in reaction with Klenow using anequimolar amount of the oligonucleotide primer 5′ATC ACA GCA CGC AGA TCTATG 3′ were used (SEQ ID NO:60). After extension, the resulting dsDNAwas digested with Bgl II and ligated into the pLAC11 expression vectorwhich had been digested with the same restriction enzyme andsubsequently dephosphorylated using alkaline phosphatase. Because of theway the oligonucleotide library has been engineered, either orientationof the incoming digested double-stranded DNA fragment results in afusion product.

[0170] To construct the randomized peptide libraries for use with thep(N)Rop fusion vector, the randomized oligonucleotide 5′TAC TAT GAA TTC(NNN)₂₀ GAA TTC TGC CAC CAC TAC TAT 3′ (SEQ ID NO:61), and the primer5′ATA GTA GTG GTG GCA GAA TTC 3′ (SEQ ID NO:62) were used. Afterextension, the resulting dsDNA was digested with EcoRI and ligated intothe pLAC11 expression vector which had been digested with the samerestriction enzyme and subsequently dephosphorylated using alkalinephosphatase. Because of the way the oligonucleotide library has beenengineered, either orientation of the incoming digested double-strandedDNA fragment results in a fusion product.

[0171] Generation of a Randomized Peptide Library Containing TerminalProlines. Randomized amino acid peptide libraries containing two prolineresidues at both the amino and the carboxy terminal ends of the peptideswere constructed using the synthetic oligonucleotide 5′TAC TAT AGA TCTATG CCG CCG (NNN)₁₆ CCG CCG TAA TAA GAA TTC GTA CAT 3′ (SEQ ID NO:63).The complementary strand of the 93 base randomized oligonucleotide wasgenerated by filling in with Klenow using the oligonucleotide primer5′ATG TAC GAA TTC TTA TTA CGG CGG 3′ (SEQ ID NO:64). After extension,the resulting dsDNA was digested with Bgl II and EcoR I and ligated intothe pLAC11 expression vector which had been digested with the same tworestriction enzymes. Because the initiating methionine of the peptidescoded by this library is followed by a proline residue, the initiatingmethionine will be removed (F. Sherman et al, Bioessays 3:27-31 (1985)).Thus the peptide libraries encoded by this scheme are 20 amino acids inlength.

[0172] Generation of a Randomized Hydrophilic α-Helical Peptide Library.Table 14 shows the genetic code highlighted to indicate certain aminoacid properties. TABLE 14 Genetic Code Highlighted to Indicate AminoAcid Properties TTT phe h_(a) TCT ser TAT tyr b_(a) TGT cys TTC pheh_(a) TCC ser TAC tyr b_(a) TGC cys TTA leu H_(a) TCA ser TAA OCH TGAOPA TTG leu H_(a) TCG ser TAG AMB TGG trp CIT leu H_(a) CCT pro B_(a)CAT his h_(a) CGT arg CTC leu H_(a) CCC pro B_(a) CAC his h_(a) CGC argCTA leu H_(a) CCA pro B_(a) CAA gin h_(a) CGA arg CTG leu H_(a) CCG proB_(a) CAG gln h_(a) CGG arg ATT ile h_(a) ACT thr AAT asn b_(a) ACT serATC ile h_(a) ACC thr AAC asn b_(a) AGC ser ATA ile h_(a) ACA thr AAAasn h_(a) AGA arg ATG met H_(a) ACG thr AAG asn h_(a) AGG arg GTT valh_(a) GCT ala H_(a) GAT asp h_(a) CGT gly B_(a) GTC val h_(a) CCC alaH_(a) GAC asp h_(a) GGC gly B_(a) GTA val h_(a) GCA ala H_(a) GAA asph_(a) GGA gly B_(a) GTG val h_(a) GCG ala H_(a) GAG asp h_(a) GGG glyB_(a) # Enzymol. 47:45-148 (1978); P. Chou, “Prediction of proteinstructural classes from amino acid compositions,” in Prediction ofprotein structure and the principles of protein conformation (C. Fasman,G. D. ed.). Plenum Press, New York, N.Y. 549-586 (1990)); Gamier,Osguthorpe, and Robson (J Mol. Bid. 120:97-120 (1978)); and O'Neill andDeCrado (Science. 250:646-65 1 (1990)) methods for predicting secondarystructure.

[0173] By analyzing the distribution pattern of single nucleotides inthe genetic code relative to the properties of the amino acids encodedby each nucleotide triplet, a novel synthetic approach was identifiedthat would yield randomized 18 amino acid hydrophilic peptide librarieswith a propensity to form α-helices. According to Table 14, the use of a[(CAG)A(TCAG)] codon mixture yields the hydrophilic amino acids His,Gln, Asn, Lys, Asp, and Glu. These amino acids are most often associatedwith α-helical motifs except for asparagine, which is classified as aweak α-helical breaker. If this codon mixture was used to build anα-helical peptide, asparagine would be expected to occur in about 17% ofthe positions, which is acceptable in an α-helical structure accordingto the secondary structure prediction rules of either Chou and Fasman(P. Chou et al., Adv. Enzymol. 47:45-148 (1978); P. Chou, “Prediction ofprotein structural classes from amino acid compositions,” in Predictionof protein structure and the principles of protein conformation (G.Fasman, G. D. ed.). Plenum Press, New York, N.Y. 549-586 (1990)) orGamier, Osguthorpe, and Robson (J. Gamier et al., J. Mol. Biol.120:97-120 (1978)). Additionally, several well-characterized proteinshave been observed to contain up to three b_(a) breaker amino acidswithin a similarly sized α-helical region of the protein (T. Creighton,“Conformational properties of polypeptide chains,” in Proteins:structures and molecular properties, W. H. Freeman and Company, N.Y.,182-186 (1993)). Since in most α-helices there are 3.6 amino acids percomplete turn, the 18 amino acid length was chosen in order to generateα-helical peptides which contained 5 complete turns. Moreover, the useof hydrophilic amino acids would be expected to yield peptides which aresoluble in the cellular cytosol.

[0174] Randomized 18 amino acid hydrophilic α-helical peptide librarieswere synthesized using the synthetic oligonucleotide 5′TAC TATAGATCTATG(VAN)₁₇ TAA TAA GAA TTC TGC CAG CAC TAT 3′ (SEQ ID NO:65). Thecomplementary strand of the 90 base randomized oligonucleotide wasgenerated by filling in with Klenow using the oligonucleotide primer5′ATA GTG CTG GCA GAA TTC TTA TTA 3′ (SEQ ID NO:66). After extension theresulting dsDNA was digested with Bgl II and EcoR I and ligated into thepLAC11 expression vector which had been digested with the same tworestriction enzymes.

[0175] Generating a Randomized Peptide Library Containing the +/− ChargeEnding Motif. Randomized peptide libraries stabilized by the interactionof oppositely charge amino acids at the amino and carboxy termini weregenerated according to the scheme shown in FIG. 9. To maximize thepotential interactions of the charged amino acids, the larger acidicamino acid glutamate was paired with the smaller basic amino acidlysine, while the smaller acidic amino acid aspartate was paired withthe larger basic amino acid arginine. To construct the randomizedpeptide libraries, the synthetic oligonucleotide 5′TAC TAT AGA TCT ATGGAA GAC GAA GAC (NNN)₁₆ CGT AAA CGT AAA TAA TAA GAA TTC GTA CAT 3′ (SEQID NO:67)and the oligonucleotide primer 5′ATG TAC GAA TTC TTA TTA TTTACG TTT ACG 3′ (SEQ ID NO: 68) were used. After extension, the resultingdsDNA was digested with Bgl II and EcoR I and ligated into the pLAC11expression vector which had been digested with the same two restrictionenzymes.

[0176] For all libraries of randomized oligonucleotides, N denotes thatan equimolar mixture of the four nucleotides A, C, G, and T was used,and V denotes that an equimolar mixture of the three nucleotides A, Cand G was used. The resulting libraries were transformed intoelectrocompetent ALS225 E. coli cells (Example I) under repressedconditions as described in Example II.

[0177] Screening of Transformants to Identify Inhibitor Clones.Transformants were initially screened using the grid-patching techniqueto identify any that could not grow on minimal media as described inExample II when the peptides were overproduced. To verify that all theinhibitors were legitimate, plasmid DNA was made from each inhibitoryclone, transformed into a fresh background, then checked to make surethat they were still inhibitory on plates and that their inhibition wasdependent on the presence of the inducer, IPTG, as in Example II.

[0178] Growth Rate Analysis in Liquid Media. Inhibition strength of thepeptides was assessed by subjecting the inhibitory clones to a growthrate analysis in liquid media. Minimal or rich cultures containingeither the inhibitor to be tested or the relevant vector as a controlwere diluted to an initial OD₅₅₀ of approximately 0.01 using new mediaand induced with 1 mM IPTG. OD₅₅₀ readings were then taken hourly untilthe cultures had passed log phase. Growth rates were determined as thespectrophotometric change in OD₅₅₀ per unit time within the log phase ofgrowth, and inhibition of the growth rate was calculated for theinhibitors using the appropriate vector as a control.

[0179] Results

[0180] Isolation and Characterization of Inhibitor Peptides that areFused at Their Carboxy Terminal End to the Amino Terminal End of the RopProtein. Approximately 10,000 peptides protected by the Rop protein attheir carboxy terminal end were screened using the grid-patchingtechnique described in Example II, and 16 two day inhibitors wereisolated. The inhibitory effects were determined as described in theExample II, using pRop(C) as a control. Unlike the anchorless inhibitorsidentified in Example II that were only inhibitory on minimal media,many of the Rop fusion inhibitors were also inhibitory on rich media aswell, which reflects increased potency. As indicated in Table 15, theinhibitors inhibited the bacterial growth rate at levels that averaged90% in minimal media and at levels that averaged 50% in rich media. Thedata in Table 15 is the average of duplicate experiments. TABLE 15Inhibitory effects of peptide inhibitors stabilized by fusing thecarboxy terminal end of the peptide to the amino terminal end of the Ropprotein (Rop(C) fusion peptide inhibitors % inhibition in % inhibitionin Inhibitor minimal media rich media pRop(C)1 87 47 pRop(C)2 99 58pRop(C)3 85 54 pRop(C)4 98 49 pRop(C)5 95 54 pRop(C)6 99 46 pRop(C)7 9159 pRop(C)8 86 51 pRop(C)9 93 57 pRop(C)10 91 35

[0181] Isolation and Characterization of Inhibitor Peptides that areFused at Their Amino Terminal End to the Carboxy Terminal End of the RopProtein. Approximately 6000 peptides protected at their amino terminalend by Rop protein were screened using the grid-patching techniquedescribed in Example II, and 14 two day inhibitors were isolated. Asobserved for the Rop fusion peptides isolated using the p-Rop(C) vector,most of the inhibitor peptides isolated using the p(N)Rop-vector wereinhibitory on rich media as well as minimal media. The inhibitors wereverified as described hereinabove and subjected to growth rate analysisusing p(N)Rop- as a control in order to determine their potency. Asindicated in Table 16, the inhibitors inhibited the bacterial growthrate at levels that averaged 90% in minimal media and at levels thataveraged 40% in rich media. The data in Table 16 is the average ofduplicate experiments. TABLE 16 Inhibitory effects of peptide inhibitorsstabilized by fusing the amino terminal end of the peptide to thecarboxy terminal end of the Rop protein (Rop(N) fusion peptideinhibitors) % inhibition in % inhibition in Inhibitor minimal media richmedia pRop(N)1 81 30 pRop(N)2 96 53 pRop(N)3 95 43 pRop(N)4 92 38pRop(N)5 99 33 pRop(N)6 93 38 pRop(N)7 87 34 pRop(N)8 91 44 pRop(N)9 9537 pRop(N)10 96 40

[0182] Isolation and Characterization of Anchorless Inhibitor PeptidesContaining Two Prolines at Both Their Amino Terminal and CarboxyTerminal Ends. Approximately 7500 peptides were screened using thegrid-patching technique described in Example II, and 12 two dayinhibitors were isolated. As indicated in Table 17, the top teninhibitors inhibited the bacterial growth rate at levels that averaged50% in minimal media. The inhibitory effects were determined asdescribed in the text using pLAC11 as a control. The data in Table 17 isthe average of duplicate experiments. TABLE 17 Inhibitory effects ofpeptide inhibitors stabilized by two proline residues at both the aminoand carboxy terminal ends of the peptide % inhibition in Inhibitorminimal media pPro1 50 pPro2 49 pPro3 50 pPro4 59 pPro5 52 pPro6 93pPro7 54 pPro8 42 pPro9 41 pPro10 42

[0183] Sequence analysis of the coding regions for the top teninhibitors is shown in Table 19. The landmark Bgl II and EcoR Irestriction sites for the insert region are underlined, as are theproline residues.

[0184] Since the ends of the oligonucleotide from which these inhibitorswere constructed contained Bgl II and EcoRI I restriction sites, theoligonucleotide was not gel isolated when the libraries were prepared inorder to maximize the oligonucleotide yields. Because of this, three ofthe inhibitory clones, pPro2, Ppro5, and pPro6 were found to containdeletions in the randomized portion of the oligonucleotide. TABLE 18Sequence analysis of the insert region from the proline peptidespPro1-21aa AGA TCT ATG CCG CCG ATT CTA TGG GGC GAA GCG AGA AAG CGC TTGTGG GGT GGG GAT CAT ACA (SEQ ID NO: 70)         M   P   P   I   L   W   G   E   A   R   K   R   L   N   G   G   D   H   T(SEQ ID NO: 69) CCG CCG TAA TAA GAA TTC  P   P   *  * pPro2-27aaAGA TCT ATG CCG CCG CCG TTG GAT ATT GTG TCG GGT ATT GAG GTA GGG GGG CATTTG TGG TGC (SEQ ID NO: 71)         M   P   P   P   L   D   T   V   S   G   I   E   V   G   G   H   L   W   C(SEQ ID NO: 72) CGC CGT ATT AAG AAT TCT CAT GTT TGA R   R   I  K   N   S   H   V   * pPro3-8aa AGA TCT ATG CCG CCG GAC AATCCG GTC CTG TGA TGA AGC GGA GGT CGA CCA AGG GGA TAT CAG (SEQ ID NO: 73)        M   P   P   D   N   P   V   L   *   * (SEQ ID NO: 74) CCG GGGTAA TAA GAA TTC pPro4-9aa AGA TCT ATG CCG CCG CTA TTG GAC GGA GAT GACAAA TAG ATA TAT GCG TGG TTG TTT TTG TGT (SEQ ID NO: 75)        M   P   P   L   L   D   G   D   D   K   * (SEQ ID NO:76) CCG CCGTAA TAA GAA TTC pPro5-10aa AGA TCT ATG CCG CCG AGG TGG AAG ATG TTG ATAAGA CAG TGA CAG ATG CGT TGG ATT ACT CCC (SEQ ID NO: 77)        M   P   P   R   W   K   M   L   I   R   Q   * (SEQ ID NO: 78)GCC GTA ATA AGA ATT C pPro6-7aa AGA TCT ATG ATG AGA GTA GCG CCG CCG TAATAA GAA TTC (SEQ ID NO: 79)         M   M   R   V   A   P   P   *   *(SEQ ID NO: 80) pPro7-14aa AGA TCT ATG GGG CCC TTG CGC GGG GCA TGC GATGTA TAT GGG GTA AAT TGA ATG TCT TGT GGG (SEQ ID NO: 81)        M   P   P   L   N   R   A   C   D   V   V   C   V   N   * (SEQID NO 82) CCG CCG TAA TAA GAA TTC pPro8-21aa AGA TCT ATG CCG CCG GGG AGAGGG GAA GCG GTG GGA GTG ACA TGC TTG AGC GCG AAC GTG TAC (SEQ ID NO: 83)        M   P   P   G   R   G   E   A   V   G   V   T   C   L   S   A   N   V   Y(SEQ ID NO: 84) CCG CCG TAA TAA GAA TTC P   P   *   * pPro9-21 aaAGA TCT ATG CCG CCG GGA AGG GTA GTG TTC TTT GTC GCT ATC TTT GTT TCC GCAATA TGC CTC (SEQ ID NO: 85)        M   P   P   G   R   V   V   F   F   V   A   F   F   V   S   A   I   C   L(SEQ ID NO: 86) CCG CCG TAA TAA GAA TTC P   P   *   * pPro10-21aaAGA TCT ATG CCG CCG AGG TTC GCT CAT GAG AGT GTT AAA GGG CTG GGG GAC GTTACA AAA GCT (SEQ ID NO: 87)        M   P   P   R   F   A   H   K   S   V   K   G   L   G   D   V   T   K   A(SEQ ID NO: 88) CCG CCG TAA TAA GAA TTC P   P   *   *

[0185] All the inhibitors were found to contain two proline residues ateither their amino or carboxy termini as expected. Four inhibitorscontained two proline residues at both their amino and. carboxy termini,five inhibitors contained two proline residues at only their aminotermini, and one inhibitor contained two proline residues at only itscarboxy terminus.

[0186] Isolation and Characterization of Anchorless HydrophilicInhibitor Peptides Stabilized by an α-Helical Motif. Approximately12,000 peptides were screened using the grid-patching technique and 5two-day inhibitors were isolated. The inhibitors were verified asalready described for the Rop-peptide fusion studies and subjected togrowth rate analysis using pLAC11 as a control in order to determinetheir potency. As indicated in Table 19, the inhibitor peptidesinhibited the bacterial growth rate at levels that averaged 50% inminimal media. The averaged values of two independent determinations areshown. TABLE 19 Inhibitor effects of the hydrophilic α-helical peptides% inhibition in Inhibitor minimal media pHelix1 67 pHelix2 46 pHelix3 48pHelix4 45 pHelix5 42

[0187] Sequence analysis of the coding regions for the 5 inhibitors isshown in Table 20. The landmark Bgl II and EcoR I restriction sites forthe insert region are underlined. Since the ends of the oligonucleotidefrom which these inhibitors were constructed contained these restrictionsites, the oligonucleotide was not gel isolated when the libraries wereprepared in order to maximize the oligonucleotide yields. Because ofthis, two of the inhibitory clones, pHelix2 and pHelix3, were found tocontain deletions in the randomized portion of the oligonucleotide. Thepredicted α-helical content of these peptides is indicated in Table 20according to the secondary structure prediction rules of Gamier,Osguthorpe, and Robson (J. Gamier et al., J. Mol. Biol. 120: 97-120(1978)) prediction rules. TABLE 20 Sequence analysis of the insertregion from the hydrophilic α-helical peptides pHelix1-18aa, 83%α-helical AGA TCT ATG CAT GAG GAA CAA GAG GAG GAG CAC AAT AAA AAG GATAAC GAA AAA GAA GAG TAA (SEQ ID NO: 89)        M   H   D   E   D   Q   E   E   E   H   N   K   K   D   N   E   K   E   H(SEQ ID NO: 90) TAA TAA TTC *   *    pHelix2-22aa, 68% α-helicalAGA TCT ATG CAG CAG GAG CAC GAG CAA GGC AGG ATG AGG AGG AGG ATG AAG AATAAT AAG AAT (SEQ ID NO: 91)        M   Q   Q   E   H   E   Q   G   R   M   S   K   R   M   K   N   N   K   N(SEQ ID NO: 92) TCT CAT GTT TGA S   H   V   * pHelix3-22aa, 55%α-helical AGA TCT ATG AAC CAT CAT AAT GAG GCC ATG ATC AAC ACA ATG AAAACG AGG AAT AAT AAG AAT (SEQ ID NO: 93)        M   N   H   H   N   E   A   M   I   N   T   M   K   T   R   N   N   K   N(SEQ ID NO: 94) TCT CAT GTT TGA S   H   V   * pHelix4-18aa, 17%α-helical AGA TCT ATG AAC GAC GAC AAT CAG CAA GAG GAT AAT CAT GAT CAGCAT AAG GAT AAC AAA TAA (SEQ ID NO: 95)        M   N   D   D   N   Q   Q   E   D   N   H   D   Q   H   K   D   N   K   *(SEQ ID NO: 96) TAA TAA TTC * pHelix5-18aa, 50% α-helical AGA TCT ATGCAA GAG CAG GAT CAG CAT AAT GAT AAC CAT CAC GAG GAT AAA CAT AAG AAG TAA(SEQ ID NO: 97)        M   Q   E   Q   D   Q   H   N   D   N   H   H   E   D   K   H   K   K   *(SEQ ID NO: 98) TAA TAA TTC *

[0188] According to Gamier, Osguthorpe, and Robson secondary structureprediction, all of the encoded peptides are expected to be largelyα-helical except for pHelix4. Interestingly, pHelix1, which had thehighest degree of α-helical content, was also the most potent inhibitorypeptide that was isolated in this study.

[0189] Isolation and Characterization of Anchorless Inhibitor PeptidesStabilized by an Opposite Charge Ending Motif. Approximately 20,000peptides were screened using the grid-patching technique and 6 two dayinhibitors were isolated. The inhibitors were verified as alreadydescribed for the Rop-peptide fusion studies and subjected to growthrate analysis using pLAC11 as a control in order to determine theirpotency. As indicated in Table 21, the inhibitor peptides inhibited thebacterial growth rate at levels that averaged 50% in minimal media. Theaveraged values of two independent determinations are shown. TABLE 21Inhibitory effects of peptide inhibitors that are stabilized by theopposite charge ending motif % inhibition in Inhibitor minimal mediap+/−1 41 p+/−2 43 p+/−3 48 p+/−4 60 p+/−5 54 p+/−6 85

[0190] Sequence analysis of the coding regions for the six inhibitors isshown in Table 22. The landmark Bgl II and EcoR I restriction sites forthe insert region are underlined. With the exception of p+/−4, which wasterminated prematurely, the coding regions for the inhibitors were asexpected based on the motif that was used to generate the peptidelibraries. TABLE 22 Sequence analysis of the insert region from theopposite charge ending peptides P+/−1-25aa AGA TCT ATG GAA GAC GAA GACGAG GGT GCG TGA GGG TGG GGA GCA GAA CTT TGG TCG TGG CAG (SEQ ID NO: 99)        M   E   D   E   D   E   G   A   S   A   W   G   A   E   L   W   S   W   Q(SEQ ID NO: 100) TCG GTG CGT AAA CGT AAA TAP TAA GGA TTCS   V   R   K   R   K p+/−2-25aa AGA TCT ATG GAA GAC GAA GAC GGT CTA GGCATG GGG GGT GGG TTG GTC AGG CTC ACT TTA TTA (SEQ ID NO: 101)        M   E   D   E   D   G   L   G   M   G   G   G   L   V   R   L   T   L   L(SEQ ID NO: 102) TTC TTC CGT AAA CGT AAA TAA TAA GAA TTCF   F   R   K   R   K   *   * P+/−3-25aa AGA TCT ATG GAA GAC GAA GAC GGGGAG AGG ATC CAG GGG GCC CGC TGT CCA GTA GCG CTG GTA (SEQ ID NO: 103)        M   E   D   E   D   G   E   R   I   Q   G   A   R   C   P   V   A   L   V(SEQ ID NO: 104) GAT AGA CGT AAA CGT AAA TAA TAA GAA TTCD   R   R   K   R   K   *   * p+/−4-11aa AGA TCT ATG GAA GAC GAA GAC GACACG GGG CGT GGG CGG TAG CTT TAA GTT GCG CTA AGT TGC (SEQ ID NO: 106)        M   E   D   E   D   D   R   G   R   G   R   * (SEQ IQ NO: 105)GAG ATA CGT AAA CGT AAA TAA TAA GAA TTC p+/−5-25aa AGA TCT ATG GAA GACGAA GAC GGG GGG GCC GGG AGG AGG GCC TGT CTT TGT TCC GCG CTT GTT (SEQ IDNO: 107)        M   E   D   E   D   G   G   A   G   R   R   A   C   L   C   S   A   L   V(SEQ ID NO: 108) GGG GAA CGT AAA CGT AAA TAA TAA GAA TTCG   E   K   K   R   K   * p+/−6-25aa AGA TCT ATG GAA GAC GAA GAC AAG CGTCGC GAG AGG AGT GCA AAA GGG CGT CAT GTC GGT CGG (SEQ ID NO: 109)        M   E   D   E   D   K   R   R   E   R   S   A   K   G   R   H   V   G   R(SEQ ID NO: 110) TCG ATG CGT AAA CGT AAA TAA GAC TGTS   M   R   K   R   K   *

[0191] Discussion

[0192] In Example II, where fully randomized peptides were screened forinhibitory effect, only three peptides (one “anchorless” and twounanticipated Rop fusions resulting from deletion) were identified outof 20,000 potential candidates as a potent (i.e., two day) inhibitor ofE. coli bacteria. Using a biased synthesis as in this Example, it waspossible to significantly increase the frequency of isolating potentgrowth inhibitors (see Table 23). TABLE 23 Summary of the frequency atwhich the different types of inhibitor peptides can be isolatedFrequency at which a two day inhibitor peptide Type of inhibitor peptidecan be isolated Reference anchorless 1 in 20,000 Example II protected atthe C-terminal 1 in 625 This example end via Rop protected at theN-terminal 1 in 429 This example end via Rop protected at both the C- 1in 625 This example terminal and N-terminal end via two prolinesprotected with an α-helix 1 in 2,400 This example structural motifprotected with an opposite 1 in 3,333 This example charge ending motif

[0193] Many more aminopeptidases have been identified thancarboxypeptidases in both prokaryotic and eukaryotic cells (J. Bai, etal., Pharm. Res. 9: 969-978 (1992); J. Brownlees et al., J. Neurochem.60:793-803 (1993); C. Miller, In Escherichia coli and Salmonellatyphimurium cellular and molecular biology, 2nd edition (Neidhardt, F.C. ed.), ASM Press, Washington, D.C. 1:938-954 (1996)). In the Ropfusion studies, it might have therefore been expected that stabilizingthe amino terminal end of the peptide would have been more effective atpreventing the action of exopeptidases than stabilizing the carboxy endof the peptides. Surprisingly, it was found that stabilizing either endof the peptide caused about the same effect.

[0194] Peptides could also be stabilized by the addition of two prolineresidues at the amino and/or carboxy termini, the incorporation pfopposite charge ending amino acids at the amino and carboxy termini, orthe use of helix-generating hydrophilic amino acids. As shown in Table23, the frequency at which potent inhibitor peptides could be isolatedincreased significantly over that of the anchorless peptidescharacterized in Example II.

[0195] These findings can be directly implemented to design moreeffective peptide drugs that are resistant to degradation by peptidases.In this example, several strategies were shown to stabilize peptides ina bacterial host. Because the aminopeptidases and carboxypeptidases thathave been characterized in prokaryotic and eukaryotic systems appear tofunction quite similarly (C. Miller, In Escherichia coli and Salmonellatyphimurium cellular and molecular biology, 2nd edition (Neidhardt, F.C. ed.), ASM Press, Washington, D.C. 1:938-954 (1996); N. Rawlings etal., Biochem J. 290: 205-218 (1993)), the incorporation of on or more ofthese motifs into new or known peptide drugs should slow or prevent theaction of exopeptidases in a eukaryotic host cell as well.

Example IV Confirmation of the Stabilizing Effects of Proline ResiduesUsing an in Vitro System

[0196] To extend the in vivo studies described above, an in vitro systemfor directly assessing peptide stability was developed. In the in vitrosystem, peptides to be tested were mixed with a cellular extractcontaining the proteases and peptidases present in a particular celltype. To validate this approach, the stability or half-life of arandomized biotinylated peptide initially was measured using bothwild-type bacterial extracts and bacterial extracts that were deficientin known proteases or peptidases.

[0197] Materials and Methods

[0198] Bacterial strains. The bacterial strains used in this study areshown in Table 24. MG1655 clpP::cam was constructed by transducingMG1655 to chloramphenicol resistance using a P1 lysate that was preparedfrom SG22098. TABLE 24 Bacterial strains Strain Genotype Reference E.coli strains MG1655 F- λ- Guyer, M.S. et al., 1980* MG1655 F- λ-lon::Tn10 Carol Gross, University of lon::Tn10 California, San FranciscoMG1655 F- λ- clpP::cam This study clpP::cam SG22098 F- λ- araD139Δ(lac)U169 rpsL150 thi Michael Maurizi, National fIbB5301 deoC7 ptsF25clpP::cam Cancer Institute S. typhimurium LT2 strains TN1379 leuBCD485Charles Miller, University of Illinois TN1727 leuBCD485 pepA16 pepB11pepN90 pepP1 Charles Miller, University of pepQ1 pepT1 ΔsupQ302(proABpepD) optA1 Illinois zxx848::Tn5 dcp-1 zxx845::Tn10

[0199] Media. Bacterial cells were grown in LB media; yeast cells weregrown in 1.0% yeast extract, 2.0% peptone, 2.0% glucose; human HeLacells (ATCC CCL-2) and colon CCD-18Co cells (ATCC CRL-1459) were grownin Minimal Essential Medium Eagle (ATCC 30-2003) with Earle's balancedsalt solution, 0.1 mM non-essential amino acids, 2.0 mM L-glutamine, 1.0mM sodium pyruvate, 1.5 g/L NaHCO₃, and 10% fetal bovine serum; andhuman small intestine FHs74 Int cells (ATCC CCL-241) were grown inHybri-Care media (ATCC 46-X) with 1.5 g/L NaHCO₃ and 10% fetal bovineserum.

[0200] Preparation of the extracts. For bacteria and yeast, 500 mL ofcells were grown to an OD₅₅₀ of 0.5, centrifuged, washed twice withT₁₀E₀ ₁ (10.0 mM Tris; pH 8.0, 0.1 mM EDTA; pH 8.0) and resuspended in2.0 mL of 10.0 mM Tris; pH 8.0. For human cells, 10-50 75 cm² T flaskswere seeded and allowed to grow to 95% confluency in a 37° C. incubatorwith 5% CO₂ atmosphere. Each flask was then washed with HBSS (0.4 g/LKCl, 0.06 g/L KH₂PO₄, 8 g/L NaCl, 0.35 g/L NaHCO₃, 0.048 g/L Na₂HPO₄,1.0 g/L Glucose) that contained 0.125 mM EDTA; pH 8.0. To liberate thecells, the flasks were treated with 1.5 mL of HBSS that contained 0.25%trypsin and 0.5 mM EDTA; pH 8.0. The trypsin was neutralized by adding 5mL of media with 10% fetal bovine serum to each flask. The cells werecentrifuged, washed with HBSS that contained 0.125 mM EDTA; pH 8.0,washed twice with HBSS lacking glucose and ETDA, and resuspended in 2.0mL of 10.0 mM Tris; pH 8.0. All cell suspensions were lysed with threepasses at 15,000 psi in a French Pressure cell maintained at 4° C. Thelysates were then centrifuged at 15,000 rpm, 4° C., for 10 minutes topellet debris and unlysed cells and the supernatant was saved as thecell extract. To prepare rat serum, one 300 g Sprague Dawley rat waseuthanized with CO₂ and a heart puncture was performed to draw the bloodwhich was immediately transferred to a tube and centrifuged at 4° C.,10,000 rpm, for 10 minutes. The cleared serum was removed with a pipetteexcept for 1 cm of serum at the interface with the blood cell pellet.

[0201] Peptide synthesis. The following randomized biotinylated peptideswere synthesized by Sigma Genosys (The Woodlands, Texas): UnprotectedXXXXXX[KBtn]XXXXXA P at both ends PXXXX[KBtn]XXXXP PP at both endsPPXXXX[KBtn]XXXXPP APP at both ends APPXXXX[KBtn]XXXXPPA APP aminoAPPXXXX[K-Btn]XXXXA APP carboxyl AXXXX[K-Btn]XXXXPPA Acetylated(Ac)AXXXXX[KBtn]XXXXXA Amidated XXXXXX[KBtn]XXXXXA(NH₂) CyclizedCXXXXXX[KBtn]XXXXXXC

[0202] where A denotes the L-amino acid alanine, P denotes the L-aminoacid proline, X denotes an equimolar mixture of the 20 natural L-aminoacids except for proline, and KBtn denotes the L-amino acid lysine towhich biotin has been attached.

[0203] To ensure that the length of the randomized portion of thepeptides did not affect the degradation profiles, we also tested theunprotected peptides XXXX[KBtn]XXXXA and AXXXX[KBtn]XXXXA. Theirhalf-lives were determined to be within 5% of the XXXXXX[KBtn]XXXXXApeptide which was used as the control for these studies.

[0204] In vitro degradation assay. All extracts were used at a finalconcentration of 10 mg/mL, except for the S. typhimurium extracts, whichwere used at a final concentration of 25 mg/mL. The cell extract (50 μL)was mixed with 50 μL of a peptide at a concentration of 1 mg/mL in 10 mMTris; pH 8.0 and incubated at 37° C. Aliquots were removed (10 μL) at30, 60, 90, or 120 minute intervals, placed into 90 μL of SDS-PAGEgradient gel buffer, boiled for 5 minutes, and electrophoresed through a10-20% tricine gradient gel. The gel was blotted onto a nitrocellulosemembrane and the resulting Western blot was treated with NeutrAvidinHorseradish Peroxidase Conjugate and SuperSignal West Dura ExtendedDuration Chemiluminescent Substrate (Pierce, Rockford, Ill.). Thebiotinylated peptides were then visualized by exposing the blots toautoradiography film and the resulting bands were quantified using theAlphaEase 5.5 Densitometry Program from Alpha Innotech, San Leandro,Calif.

[0205] Results

[0206] The proteases and peptidases have been well characterized in E.coli and S. typhimurium. In E. coli, the two main proteases that havebeen shown to have a role in peptide degradation are Lon and CIpP, whichare encoded respectively by the lon and clpP genes. In S. typhimurium,numerous peptidases have been identified, and strains have beenconstructed that delete several of the peptidases. Using extractsprepared from E. coli strains that contained lon or clpP deletions and aS. typhimurium strain in which nine peptidase genes were deleted,half-lives were determined for the unprotected randomized biotinylatedcontrol peptide. As shown in Table 25, deletion of the Lon proteasecaused the peptide's half-life to increase by 6.5 fold, deletion of theClpP protease caused the peptide's half-life to increase by 1.8 fold,and deletion of multiple peptidases caused the peptide's half-life toincrease by 7.1 fold. These results prove that the in vitro systemprovides an accurate method by which to assess peptide stability. TABLE25 Peptide degradation in protease and peptidase deficient extracts.Strain from which extract was prepared* Peptide half-life in minutesMG1655 44.9 MG1655 lon::Tn10 290.6 MG1655 clpP::cam 82.5 TN1379 42.0TN1379 dcp-1 optA1 pepA16 pepB11 298.5 ΔpepD pepN90 pepP1 pepQ1 pepT1

[0207] With the system validated, the stabilizing effects of prolineresidues were analyzed. Three randomized biotinylated peptides weretested using extracts prepared from bacterial (wild-type E. coli),Baker's yeast (wild-type Saccharomyces cerevisiae), human (HeLa) cells,human intestine and colon cells, and rat serum. One randomized peptidewas unprotected, while the other two peptides were stabilized on boththe N- and C-termini with a Pro (P) motif, a Pro-Pro motif (PP), or anAla-Pro-Pro motif (APP). The results are shown in the Table 26. TABLE 26The effect of proline-containing stabilizing groups on peptidedegradation Peptide half-lives in minutes Peptide Peptide Peptideprotected protected protected at Unprotected at both at both both endsby Extract peptide ends by P ends by PP APP E. coli 44.9 38.2 51.1 69.8S. cereviseae 23.3 44.4 99.0 156.0 Human HeLa 90.8 ND 423.4 1,054.3Human 121.6 99.2 166.3 171.8 Intestine Human 58.1 64.5 76.1 109.2 ColonRat serum 54.1 80.7 85.3 154.5

[0208] As the data indicate, the APP motif offered significantly moreprotection than the PP motif, which provided better protection than theP motif.

[0209] Table 27 shows the results of degradation studies on peptidesthat contain the APP motif at either or both of the amino or carboxyltermini. TABLE 27 The effect of APP stabilizing groups on peptidedegradation Peptide half-lives in minutes APP APP at both APP AminoCarboxyl Extract Unprotected ends terminus terminus E. coli 44.9 69.899.6 54.6 S. cereviseae 23.3 156.0 86.0 44.4 Human Intestine 121.6 171.8200.7 99.0 Human Colon 58.1 109.2 144.0 95.1 Rat serum 54.1 154.5 165.3121.2

[0210] The data show that APP at only the amino terminus offers slightlybetter protection than APP at both termini, and that APP at only theamino terminus offers significantly better protection than APP at onlythe carboxyl terminus.

[0211] Table 28 shows the results of degradation studies on peptidesthat contain the APP motif at the N- or C-terminus compared to peptidesthat are acetylated at their amino terminus, amidated at their carboxylterminus, or cyclized. TABLE 28 The effect of APP stabilizing groups onpeptide degradation in comparison to acetylation, amidation orcyclization. Peptide half-lives in minutes Extract Unprotected APP AminoAcetylated APP Carboxyl Amidated Cyclized E. coli 44.9 99.6 34.9 54.646.7 52.3 S. cereviseae 23.3 86.0 44.2 44.4 73.9 145.0 Rat serum 54.1165.3 67.3 121.2 75.7 217.2

[0212] The data clearly shows that APP at the amino terminus offersbetter protection than amidating the carboxyl terminus or acetylatingthe amino terminus, and is almost as good as cyclization.

Example V Bioactivity of Natural Galanin, APP-galanin, andAPP-galanin-PPA

[0213] Radio immunoassays (RIAs) were performed to determine the abilityof galanin and its APP derivatives to displace radiolabeled galanin fromits receptor. Binding (displacement) constants were then calculated fromthis data. Natural galanin Ki = 5.21 × 10⁻⁹ APP-galanin Ki = 6.42 × 10⁻⁹APP-galanin-PPA Ki = 9.46 × 10⁻⁹

[0214] As the data shows the binding constants for the APP derivativeswere in the same range as natural galanin and thus these compounds wereable to interact with the galanin receptor in a manner similar tonatural galanin.

Example VI In Vivo Glucagon, APP-Glucagon and APP-Glucagon-PPADegradation

[0215] A catheter was placed in the right jugular vein of six MaleSprague-Dawley rats for dosing and sampling. Two rats were used for eachof the three compounds that were tested. The rats received anintravenous bolus injection of the peptide, and serial blood samples(0.3 ml) were obtained. The glucagon was extracted from plasma byorganic protein precipitation and quantified by electrospray LC-MS.

[0216] The presence of the APP motif affected both the half-life ofglucagon as well as the rate at which it is cleared from the body. Thedata (Table 29) suggests that a significant portion of the glucagonharboring the APP motif becomes sequestered and thus is much moreresistant to degradation. It should be noted that significantly moreAPP-glucagon-PPA and APP-glucagon is present at 20 and 60 minutes thanwould be predicted due to its half-life. TABLE 29 Half-life Percentremaining Percent remaining Peptide in minutes after 20 minutes after 60minutes Glucagon 1.031 0.2 0.0 APP-Glucagon-PPA 1.555 3.0 NDAPP-Glucagon 2.253 7.3 8.6

[0217] The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claim.

1 115 1 133 DNA Escherichia coli 1 ggcagtgagc gcaacgcaat taatgtgagttagctcactc attaggcacc ccaggcttta 60 cactttatgc ttccggctcg tatgttgtgtggaattgtga gcggataaca atttcacaca 120 ggaaacagct atg 133 2 25 PRTARTIFICIAL peptide having opposite charge ending motif 2 Met Glu Asp GluAsp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa XaaXaa Xaa Arg Lys Arg Lys 20 25 3 14 PRT ARTIFICIAL stabilized angiotesin3 Pro Pro Asp Arg Val Tyr Ile His Pro Phe His Ile Pro Pro 1 5 10 4 18PRT ARTIFICIAL stabilized angiotensin 4 Glu Asp Glu Asp Asp Arg Val TyrIle His Pro Phe His Ile Arg Lys 1 5 10 15 Arg Lys 5 10 PRT Homo Sapiens5 Asp Arg Val Tyr Ile His Pro Phe His Ile 1 5 10 6 20 DNA ARTIFICIALprimer 6 gttgccattg ctgcaggcat 20 7 42 DNA ARTIFICIAL primer 7attgaattca taagatcttt cctgtgtgaa attgttatcc gc 42 8 37 DNA ARTIFICIALprimer 8 attgaattca ccatggacac catcgaatgg tgcaaaa 37 9 19 DNA ARTIFICIALprimer 9 gttgttgcca ttgctgcag 19 10 43 DNA ARTIFICIAL primer 10tgtatgaatt cccgggtacc atggttgaag acgaaagggc ctc 43 11 36 DNA ARTIFICIALprimer 11 tactatagat ctatgaccat gattacggat tcactg 36 12 36 DNAARTIFICIAL primer 12 tacataaagc ttggcctgcc cggttattat tatttt 36 13 47DNA ARTIFICIAL primer 13 tatcatctgc agaggaaaca gctatgacca tgattacggattcactg 47 14 47 DNA ARTIFICIAL primer 14 tacatactcg agcaggaaagcttggcctgc ccggttatta ttatttt 47 15 47 DNA ARTIFICIAL primer 15tatcatggat ccaggaaaca gctatgacca tgattacgga ttcactg 47 16 36 DNAARTIFICIAL primer 16 tactatagat ctatggctat cgacgaaaac aaacag 36 17 40DNA ARTIFICIAL primer 17 atatataagc ttttaaaaat cttcgttagt ttctgctacg 4018 35 DNA ARTIFICIAL primer 18 tactatagat ctatgaacaa aggtgtaatg cgacc 3519 35 DNA ARTIFICIAL primer 19 attagtgaat tcgcacaatc tctgcaataa gtcgt 3520 15 DNA ARTIFICIAL primer fragment 20 agatcttatg aattc 15 21 15 DNAARTIFICIAL primer fragment 21 agatcttatg aattc 15 22 15 DNA ARTIFICIALprimer fragment 22 agatcttatg aattc 15 23 93 DNA ARTIFICIAL randomizedoligonucleotide 23 tactatagat ctatgnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnntaata agaattctcg aca 93 24 18DNA ARTIFICIAL primer 24 tgtcgagaat tcttatta 18 25 20 DNA ARTIFICIALprimer 25 tcattaatgc agctggcacg 20 26 20 DNA ARTIFICIAL primer 26ttcatacacg gtgcctgact 20 27 20 DNA ARTIFICIAL primer 27 tagctcactcattaggcacc 20 28 20 DNA ARTIFICIAL primer 28 gatgacgatg agcgcattgt 20 2992 DNA ARTIFICIAL antisense oligonucleotide 29 tactatagat ctacggtcactgaattttgt ggcttgttgg accaactgcc ttagtaatag 60 tggaaggctg aaattaataagaattctcga ca 92 30 91 DNA ARTIFICIAL antisense oligonucleotide 30tactatagat ctacgtggcg ggactcatgg attaagggta gggacgtggg gtttatgggt 60taaaatagtt tgataataag aattctcgac a 91 31 92 DNA ARTIFICIAL antisenseoligonucleotide 31 tactatagat ctacgaacgg ccgaaccaaa cgaatccgggacccaccagc cgcctaaaca 60 gctaccagct gtggtaataa gaattctcga ca 92 32 93DNA ARTIFICIAL antisense oligonucleotide 32 tactatagat ctacggaccgtgaagtgatg tgtgcggcaa aacaggaatg gaaggaacga 60 acgccatagg ccgcgtaataagaattctcg aca 93 33 93 DNA ARTIFICIAL antisense oligonucleotide 33tactatagat ctacgagggg cgccaactaa ggggggggga aggtatttgt cccgtgcata 60atctcgggtg ttgtctaata agaattctcg aca 93 34 13 PRT ARTIFICIAL stabilizedpeptide 34 Met Val Thr Glu Phe Cys Gly Leu Leu Asp Gln Leu Pro 1 5 10 3586 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 35 caggaaagatctatggtcac tgaattttgt ggcttgttgg accaactgcc ttagtaatag 60 tggaaggctgaaattaataa gaattc 86 36 16 PRT ARTIFICIAL stabilized peptide 36 Met TrpArg Asp Ser Trp Ile Lys Gly Arg Asp Val Gly Phe Met Gly 1 5 10 15 37 85DNA ARTIFICIAL nucleic acid encoding stabilized peptide 37 caggaaagatctatgtggcg ggactcatgg attaagggta gggacgtggg gtttatgggt 60 taaaatagtttgataataag aattc 85 38 141 DNA ARTIFICIAL nucleic acid encodingstabilized peptide 38 caggaaagat ctatgtcagg gggacatgtg acgagggagtgcaagtcggc gatgtccaat 60 cgttggatct acgtaataag aattctcatg tttgacagcttatcatcgat aagctttaat 120 gcggtagttt atcacagtta a 141 39 42 PRTARTIFICIAL stabilized peptide 39 Met Ser Gly Gly His Val Thr Arg Glu CysLys Ser Ala Met Ser Asn 1 5 10 15 Arg Trp Ile Tyr Val Ile Arg Ile LeuMet Phe Asp Ser Leu Ser Ser 20 25 30 Ile Ser Phe Asn Ala Val Val Tyr HisSer 35 40 40 6 PRT ARTIFICIAL stabilized peptide 40 Met Tyr Leu Phe IleGly 1 5 41 75 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 41caggaaagat ctatgtattt gttcatcgga taatacttaa tggtccgctg gagaacttca 60gtttaataag aattc 75 42 87 DNA ARTIFICIAL nucleic acid encodingstabilized peptide 42 caggaaagat ctatgcttct atttgggggg gactgcgggcagaaagccgg atactttact 60 gtgctaccgt caaggtaata agaattc 87 43 20 PRTARTIFICIAL stabilized peptide 43 Met Leu Leu Phe Gly Gly Asp Cys Gly LysAla Gly Tyr Phe Thr Val 1 5 10 15 Leu Pro Ser Arg 20 44 75 DNAARTIFICIAL nucleic acid encoding stabilized peptide 44 caggaaagatctatgattgg gggatcgttg agcttcgcct gggcaatagt ttgtaataag 60 aattctcatgtttga 75 45 20 PRT ARTIFICIAL stabilized peptide 45 Met Ile Gly Gly SerLeu Ser Phe Ala Trp Ala Ile Val Cys Asn Lys 1 5 10 15 Asn Ser His Val 2046 14 PRT ARTIFICIAL stabilized peptide 46 Met Asn Gly Arg Thr Lys ArgIle Arg Asp Pro Pro Ala Ala 1 5 10 47 86 DNA ARTIFICIAL nucleic acidencoding stabilized peptide 47 caggaaagat ctatgaacgg ccgaaccaaacgaatccggg acccaccagc cgcctaaaca 60 gctaccagct gtggtaataa gaattc 86 4818 PRT ARTIFICIAL stabilized peptide 48 Met Asp Arg Glu Val Met Cys AlaAla Lys Gln Glu Trp Lys Glu Arg 1 5 10 15 Thr Pro 49 87 DNA ARTIFICIALnucleic acid encoding stabilized peptide 49 caggaaagat ctatggaccgtgaagtgatg tgtgcggcaa aacaggaatg gaaggaacga 60 acgccatagg ccgcgtaataagaattc 87 50 87 DNA ARTIFICIAL nucleic acid encoding stabilized peptide50 caggaaagat ctatgtagcc caatgcactg ggagcacgcg tgttaggtct agaagccacg 60tacccattta atccataata agaattc 87 51 12 PRT ARTIFICIAL stabilized peptide51 Met Leu Gly Leu Glu Ala Thr Tyr Pro Phe Asn Pro 1 5 10 52 5 PRTARTIFICIAL stabilized peptide 52 Met Arg Gly Ala Asn 1 5 53 87 DNAARTIFICIAL nucleic acid encoding stabilized peptide 53 caggaaagatctatgagggg cgccaactaa ggggggggga aggtatttgt cccgtgcata 60 atctcgggtgttgtctaata agaattc 87 54 4 PRT ARTIFICIAL N-terminal protective sequence54 Xaa Pro Pro Xaa 1 55 36 DNA ARTIFICIAL primer 55 tactatagatctatgaccaa acaggaaaaa accgcc 36 56 36 DNA ARTIFICIAL primer 56tatacgtatt cagttgctca catgttcttt cctgcg 36 57 41 DNA ARTIFICIAL primer57 aattcatact atagatctat gaccaaacag gaaaaaaccg c 41 58 42 DNA ARTIFICIALprimer 58 tatataatac atgtcagaat tcgaggtttt caccgtcatc ac 42 59 96 DNAARTIFICIAL randomized oligonucleotide 59 tactatagat ctatgnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnncatagatctgcgtgc tgtgat 96 60 21 DNA ARTIFICIAL primer 60 atcacagcacgcagatctat g 21 61 36 DNA ARTIFICIAL randomized oligonucleotide 61tactatgaat tcnnngaatt ctgccaccac tactat 36 62 21 DNA ARTIFICIAL primer62 atagtagtgg tggcagaatt c 21 63 105 DNA ARTIFICIAL randomizedoligonucleotide 63 tactatagat ctatgccgcc gnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnnnnnnnnn nccgccgtaa taagaattcgtacat 105 64 24 DNA ARTIFICIAL primer 64 atgtacgaat tcttattacg gcgg 2465 90 DNA ARTIFICIAL randomized oligonucleotide 65 tactatagat ctatgvanvanvanvanvan vanvanvanv anvanvanva nvanvanvan 60 vanvantaat aagaattctgccagcactat 90 66 24 DNA ARTIFICIAL primer 66 atagtgctgg cagaattctt atta24 67 105 DNA ARTIFICIAL randomized oligonucleotide 67 tactatagatctatggaaga cgaagacnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnnnnnnncgtaa acgtaaataa taagaattcg tacat 105 68 30 DNA ARTIFICIAL primer68 atgtacgaat tcttattatt tacgtttacg 30 69 81 DNA ARTIFICIAL nucleic acidencoding stabilized peptide 69 agatctatgc cgccgattct atggggcgaagcgagaaagc gcttgtgggg tggggatcat 60 acaccgccgt aataagaatt c 81 70 21 PRTARTIFICIAL stabilized peptide 70 Met Pro Pro Ile Leu Trp Gly Glu Ala ArgLys Arg Leu Trp Gly Gly 1 5 10 15 Asp His Thr Pro Pro 20 71 90 DNAARTIFICIAL nucleic acid encoding stabilized peptide 71 agatctatgccgccgccgtt ggatattgtg tcgggtattg aggtaggggg gcatttgtgg 60 tgccgccgtattaagaattc tcatgtttga 90 72 27 PRT ARTIFICIAL stabilized peptide 72 MetPro Pro Pro Leu Asp Ile Val Ser Gly Ile Glu Val Gly Gly His 1 5 10 15Leu Trp Cys Arg Arg Ile Lys Asn Ser His Val 20 25 73 81 DNA ARTIFICIALnucleic acid encoding stabilized peptide 73 agatctatgc cgccggacaatccggtcctg tgatgaagcg gaggtcgacc aaggggatat 60 cagccgccgt aataagaatt c81 74 8 PRT ARTIFICIAL stabilized peptide 74 Met Pro Pro Asp Asn Pro ValLeu 1 5 75 81 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 75agatctatgc cgccgctatt ggacggagat gacaaataga tatatgcgtg gttgtttttc 60tgtccgccgt aataagaatt c 81 76 10 PRT ARTIFICIAL stabilized peptide 76Met Pro Pro Leu Leu Asp Gly Asp Asp Lys 1 5 10 77 79 DNA ARTIFICIALnucleic acid encoding stabilized peptide 77 agatctatgc cgccgaggtggaagatgttg ataagacagt gacagatgcg ttccattact 60 cccgccgtaa taagaattc 7978 11 PRT ARTIFICIAL stabilized peptide 78 Met Pro Pro Arg Trp Lys MetLeu Ile Arg Gln 1 5 10 79 39 DNA ARTIFICIAL nucleic acid encodingstabilized peptide 79 agatctatga tgagagtagc gccgccgtaa taagaattc 39 80 7PRT ARTIFICIAL stabilized peptide 80 Met Met Arg Val Ala Pro Pro 1 5 8181 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 81 agatctatgccgccgttgcg cggggcatgc gatgtatatg gggtaaattg aatgtcttgt 60 gggccgccgtaataagaatt c 81 82 14 PRT ARTIFICIAL stabilized peptide 82 Met Pro ProLeu Arg Gly Ala Cys Asp Val Tyr Gly Val Asn 1 5 10 83 81 DNA ARTIFICIALnucleic acid encoding stabilized peptide 83 agatctatgc cgccggggagaggggaagcg gtgggagtga catgcttgag cgcgaacgtg 60 tacccgccgt aataagaatt c81 84 21 PRT ARTIFICIAL stabilized peptide 84 Met Pro Pro Gly Arg GlyGlu Ala Val Gly Val Thr Cys Leu Ser Ala 1 5 10 15 Asn Val Tyr Pro Pro 2085 81 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 85agatctatgc cgccgggaag ggtagtgttc tttgtcgcta tctttgtttc cgcaatatgc 60ctcccgccgt aataagaatt c 81 86 21 PRT ARTIFICIAL stabilized peptide 86Met Pro Pro Gly Arg Val Val Phe Phe Val Ala Ile Phe Val Ser Ala 1 5 1015 Ile Cys Leu Pro Pro 20 87 81 DNA ARTIFICIAL nucleic acid encodingstabilized peptide 87 agatctatgc cgccgaggtt cgctcatgag agtgttaaagggctggggga cgttacaaaa 60 gctccgccgt aataagaatt c 81 88 21 PRT ARTIFICIALstabilized peptide 88 Met Pro Pro Arg Phe Ala His Glu Ser Val Lys GlyLeu Gly Asp Val 1 5 10 15 Thr Lys Ala Pro Pro 20 89 72 DNA ARTIFICIALnucleic acid encoding stabilized peptide 89 agatctatgc atgacgaacaagaggaggag cacaataaaa aggataacga aaaagaacac 60 taataagaat tc 72 90 18PRT ARTIFICIAL stabilized peptide 90 Met His Asp Glu Gln Glu Glu Glu HisAsn Lys Lys Asp Asn Glu Lys 1 5 10 15 Glu His 91 75 DNA ARTIFICIALnucleic acid encoding stabilized peptide 91 agatctatgc agcaggagcacgagcaaggc aggatgagca agaggatgaa gaataataag 60 aattctcatg tttga 75 92 22PRT ARTIFICIAL stabilized peptide 92 Met Gln Gln Glu His Glu Gln Gly ArgMet Ser Lys Arg Met Lys Asn 1 5 10 15 Asn Lys Asn Ser His Val 20 93 75DNA ARTIFICIAL nucleic acid encoding stabilized peptide 93 agatctatgaaccatcataa tgaggccatg atcaacacaa tgaaaacgag gaataataag 60 aattctcatgtttga 75 94 22 PRT ARTIFICIAL stabilized peptide 94 Met Asn His His AsnGlu Ala Met Ile Asn Thr Met Lys Thr Arg Asn 1 5 10 15 Asn Lys Asn SerHis Val 20 95 72 DNA ARTIFICIAL nucleic acid encoding stabilized peptide95 agatctatga acgacgacaa tcagcaagag gataatcatg atcagcataa ggataacaaa 60taataagaat tc 72 96 18 PRT ARTIFICIAL stabilized peptide 96 Met Asn AspAsp Asn Gln Gln Glu Asp Asn His Asp Gln His Lys Asp 1 5 10 15 Asn Lys 9772 DNA ARTIFICIAL nucleic acid encoding stabilized peptide 97 agatctatgcaagagcagga tcagcataat gataaccatc acgaggataa acataagaag 60 taataagaat tc72 98 18 PRT ARTIFICIAL stabilized peptide 98 Met Gln Glu Gln Asp GlnHis Asn Asp Asn His His Glu Asp Lys His 1 5 10 15 Lys Lys 99 93 DNAARTIFICIAL nucleic acid encoding stabilized peptide 99 agatctatggaagacgaaga cgagggtgcg tcagcgtggg gagcagaact ttggtcgtgg 60 cagtcggtgcgtaaacgtaa ataataagaa ttc 93 100 25 PRT ARTIFICIAL stabilized peptide100 Met Glu Asp Glu Asp Glu Gly Ala Ser Ala Trp Gly Ala Glu Leu Trp 1 510 15 Ser Trp Gln Ser Val Arg Lys Arg Lys 20 25 101 93 DNA ARTIFICIALnucleic acid encoding stabilized peptide 101 agatctatgg aagacgaagacggtctaggc atggggggtg ggttggtcag gctcacttta 60 ttattcttcc gtaaacgtaaataataagaa ttc 93 102 25 PRT ARTIFICIAL stabilized peptide 102 Met GluAsp Glu Asp Gly Leu Gly Met Gly Gly Gly Leu Val Arg Leu 1 5 10 15 ThrLeu Leu Phe Phe Arg Lys Arg Lys 20 25 103 93 DNA ARTIFICIAL nucleic acidencoding stabilized peptide 103 agatctatgg aagacgaaga cggggagaggatccaggggg cccgctgtcc agtagcgctg 60 gtagatagac gtaaacgtaa ataataagaa ttc93 104 25 PRT ARTIFICIAL stabilized peptide 104 Met Glu Asp Glu Asp GlyGlu Arg Ile Gln Gly Ala Arg Cys Pro Val 1 5 10 15 Ala Leu Val Asp ArgArg Lys Arg Lys 20 25 105 11 PRT ARTIFICIAL stabilized peptide 105 MetGlu Asp Glu Asp Asp Arg Gly Arg Gly Arg 1 5 10 106 93 DNA ARTIFICIALnucleic acid encoding stabilized peptide 106 agatctatgg aagacgaagacgacaggggg cgtgggcggt agctttaagt tgcgctaagt 60 tgcgagatac gtaaacgtaaataataagaa ttc 93 107 93 DNA ARTIFICIAL nucleic acid encoding stabilizedpeptide 107 agatctatgg aagacgaaga cgggggggcc gggaggaggg cctgtctttgttccgcgctt 60 gttggggaac gtaaacgtaa ataataagaa ttc 93 108 25 PRTARTIFICIAL stabilized peptide 108 Met Glu Asp Glu Asp Gly Gly Ala GlyArg Arg Ala Cys Leu Cys Ser 1 5 10 15 Ala Leu Val Gly Glu Arg Lys ArgLys 20 25 109 90 DNA ARTIFICIAL nucleic acid encoding stabilized peptide109 agatctatgg aagacgaaga caagcgtcgc gagaggagtg caaaagggcg tcatgtcggt 60cggtcgatgc gtaaacgtaa ataagactgt 90 110 25 PRT ARTIFICIAL stabilizedpeptide 110 Met Glu Asp Glu Asp Lys Arg Arg Glu Arg Ser Ala Lys Gly ArgHis 1 5 10 15 Val Gly Arg Ser Met Arg Lys Arg Lys 20 25 111 17 PRTARTIFICIAL a-helical moiety 111 Asp Trp Leu Lys Ala Arg Val Glu Gln GluLeu Gln Ala Leu Glu Ala 1 5 10 15 Arg 112 20 PRT ARTIFICIAL peptide fromneuropeptide Y 112 Ala Glu Asp Leu Ala Arg Tyr Tyr Ser Ala Leu Arg HisTyr Ile Asn 1 5 10 15 Leu Ile Thr Arg 20 113 21 PRT ARTIFICIAL peptidefrom human mannose binding protein 113 Ala Ala Ser Glu Arg Lys Ala LeuGln Thr Glu Met Ala Arg Ile Lys 1 5 10 15 Lys Ala Leu Thr Ala 20 114 24PRT ARTIFICIAL peptide from helodermin 114 Ala Ile Phe Thr Glu Glu TyrSer Lys Leu Leu Ala Lys Leu Ala Leu 1 5 10 15 Gln Lys Tyr Leu Ala SerIle Leu 20 115 34 PRT ARTIFICIAL peptide from ribosomal protein L9 115Pro Ala Asn Leu Lys Ala Leu Glu Ala Gln Lys Gln Lys Glu Gln Arg 1 5 1015 Gln Ala Ala Glu Glu Leu Ala Asn Ala Lys Lys Leu Lys Glu Gln Leu 20 2530 Glu Lys

What is claimed is:
 1. A polypeptide comprising a bioactive peptide anda first stabilizing group coupled to a terminus of said bioactivepeptide, wherein said first stabilizing group is heterologous to thebioactive peptide and lacks the capacity to participate in the formationof an intramolecular disulfide bond within the polypeptide.
 2. Thepolypeptide of claim 1, wherein said polypeptide further comprises asecond stabilizing group coupled to the other terminus of said bioactivepeptide, wherein the second stabilizing group is heterologous to thebioactive peptide.
 3. The polypeptide of claim 1, wherein said firststabilizing group is coupled to the N-terminus of said bioactivepeptide.
 4. The polypeptide of claim 1, wherein said first stabilizinggroup is coupled to the C-terminus of said bioactive peptide.
 5. Thepolypeptide of claim 2, wherein said first and said second stabilizinggroups are the same.
 6. The polypeptide of claim 1, wherein said firststabilizing group is coupled to said bioactive peptide via a peptidebond.
 7. The polypeptide of claim 2, wherein said first and said secondstabilizing groups are coupled to said bioactive peptide via peptidebonds.
 8. The polypeptide of claim 1, wherein said bioactive peptide isselected from the group consisting of insulin, glucagon, calcitonin,somatostatin, gonadotrophin, and secretin.
 9. The polypeptide of claim1, wherein said first stabilizing group is a single α-helix.
 10. Thepolypeptide of claim 1, wherein said first stabilizing group is atwo-helix bundle.
 11. The polypeptide of claim 1, wherein said firststabilizing group is a three-helix bundle.
 12. The polypeptide of claim1, wherein said first stabilizing group is a four-helix bundle.
 13. Thepolypeptide of claim 1, wherein said first stabilizing group is afive-helix bundle.
 14. The polypeptide of claim 12, wherein saidfour-helix bundle is a Rop polypeptide.
 15. The polypeptide of claim 1,wherein said first stabilizing group is Xaa-Pro-Pro- or -Pro-Pro-Xaa,wherein Xaa is any amino acid.
 16. The polypeptide of claim 15, whereinXaa is Ala.
 17. The polypeptide of claim 1, wherein said bioactivepeptide is 5 to 20 amino acids in length.
 18. The polypeptide of claim2, wherein said second stabilizing group is a single α-helix.
 19. Thepolypeptide of claim 2, wherein said second stabilizing group is atwo-helix bundle.
 20. The polypeptide of claim 2, wherein said secondstabilizing group is a three-helix bundle.
 21. The polypeptide of claim2, wherein said second stabilizing group is a four-helix bundle.
 22. Thepolypeptide of claim 2, wherein said second stabilizing group is afive-helix bundle.
 23. The polypeptide of claim 21, wherein saidfour-helix bundle is a Rop polypeptide.
 24. The polypeptide of claim 2,wherein said second stabilizing group is Xaa-Pro-Pro- or -Pro-Pro-Xaa,wherein Xaa is any amino acid.
 25. The polypeptide of claim 24, whereinXaa is Ala.
 26. A polypeptide comprising a bioactive peptide and astabilizing group coupled to a terminus of said bioactive peptide,wherein said stabilizing group is not a thioredoxin polypeptide.
 27. Apolypeptide comprising a bioactive peptide, a first stabilizing groupcoupled to the N-terminus of said bioactive peptide and a secondstabilizing group coupled to the C-terminus of said bioactive peptide,wherein said first and second stabilizing groups are heterologous to thebioactive peptide and to each other.
 28. A polypeptide comprising abioactive peptide, a first stabilizing group coupled to the N-terminusof said bioactive peptide and a second stabilizing group coupled to theC-terminus of said bioactive peptide, wherein said first and secondstabilizing groups are heterologous to the bioactive peptide and do notinteract to form a naturally occurring secondary or tertiary structure.29. A polypeptide comprising a bioactive peptide, a first stabilizinggroup coupled to the N-terminus of said bioactive peptide and a secondstabilizing group coupled to the C-terminus of said bioactive peptide,wherein said first stabilizing group and second groups are heterologousto the bioactive peptide and do not confine the N-terminus and theC-terminus of the bioactive peptide in close proximity.
 30. A method ofmaking a stabilized polypeptide, said method comprising coupling astabilizing group to at least one terminus of a bioactive peptide toproduce said stabilized polypeptide, wherein said first stabilizinggroup is heterologous to the bioactive peptide and lacks the capacity toparticipate in the formation of an intramolecular disulfide bond withinthe polypeptide.
 31. The method of claim 30, wherein said coupling stepcomprises chemically synthesizing said stabilized polypeptide.
 32. Themethod of claim 30, further comprising expressing said stabilizedpolypeptide in a host cell transformed with a vector, said vectorcomprising an expression control sequence operably linked to a nucleicacid sequence encoding said stabilized polypeptide, wherein saidexpression control sequence is tightly regulable in said host cell. 33.The method of claim 32, wherein said method further comprisesdetermining stability of said stabilized polypeptide relative to saidbioactive peptide.
 34. A method of making a stabilized polypeptide, saidmethod comprising coupling a heterologous stabilizing group to at leastone terminus of a bioactive peptide to produce said stabilizedpolypeptide; and determining stability of said stabilized polypeptiderelative to said bioactive peptide.
 35. An isolated nucleic acidencoding a stabilized polypeptide, wherein said stabilized polypeptidecomprises a bioactive peptide and a first stabilizing group coupled toone of said bioactive peptide's termini, wherein said first stabilizinggroup is heterologous to the bioactive peptide and lacks the capacity toparticipate in the formation of an intramolecular disulfide bond withinthe polypeptide.
 36. The isolated nucleic acid of claim 35, wherein saidstabilized polypeptide further comprises a second stabilizing groupcoupled to the other terminus of said bioactive peptide, wherein thesecond stabilizing group is heterologous to the bioactive peptide. 37.The isolated nucleic acid of claim 35, wherein said first and saidsecond stabilizing groups are the same.
 38. A method of making astabilized polypeptide, said method comprising: a) providing host cellstransformed with an exogenous nucleic acid encoding said stabilizedpolypeptide, said stabilized polypeptide comprising a bioactive peptideand a first stabilizing group coupled to one of said bioactive peptide'stermini, wherein said first stabilizing group is heterologous to thebioactive peptide and lacks the capacity to participate in the formationof an intramolecular disulfide bond within the polypeptide; b)expressing said stabilized polypeptide; and c) recovering saidstabilized polypeptide.
 39. The method of claim 38, wherein said hostcells are bacteria.
 40. The method of claim 38, wherein said host cellsare eukaryotic host cells.
 41. The method of claim 38, wherein saidstabilized polypeptide further comprises a second stabilizing groupcoupled to the other terminus of said bioactive peptide, wherein thesecond stabilizing group is heterologous to the bioactive peptide. 42.The method of claim 41, wherein said first and said second heterologousstabilizing groups are the same.
 43. A method of making a polypeptidecomprising: providing a bacteriophage that comprises an exogenousnucleic acid encoding a polypeptide comprising a bioactive peptide, abacteriophage protein coupled to one terminus of said bioactive peptide,and a stabilizing group coupled to the other terminus of said bioactivepeptide; and culturing said bacteriophage under conditions to cause thebacteriophage to express said polypeptide and display it on the surfaceof the bacteriophage.
 44. The method of claim 43 wherein the stabilizinggroup is coupled to the N-terminus of the bioactive peptide.
 45. Themethod of claim 43 wherein the stabilizing group is coupled to theC-terminus of the bioactive peptide.
 46. The method of claim 43 furthercomprising cleaving the polypeptide from the host cell surface to yielda stabilized bioactive peptide comprising the bioactive peptide and thestabilizing group.
 47. A method of making a stabilized polypeptidecomprising coupling a stabilizing group to at a terminus of a bioactivepeptide to produce the stabilized polypeptide, said bioactive peptidehaving been identified using a phage display process that produces abacteriophage protein coupled to one terminus of said bioactive peptide,wherein the stabilizing group takes the place of the bacteriophageprotein.
 48. The method of claim 47, wherein said coupling stepcomprises chemically synthesizing said stabilized bioactive peptide. 49.The method of claim 47, further comprising expressing said stabilizedpolypeptide in a host cell transformed with a vector, said vectorcomprising an expression control sequence operably linked to a nucleicacid sequence encoding said stabilized polypeptide, wherein saidexpression control sequence is tightly regulable in said host cell. 50.The method of claim 47, further comprising identifying said bioactivepeptide using a phage display process.
 51. A polypeptide comprising abioactive peptide and a first stabilizing group coupled to a terminus ofsaid bioactive peptide, said bioactive peptide having been identifiedusing a phage display process that produces a bacteriophage proteincoupled to one terminus of said bioactive peptide, wherein thestabilizing group takes the place of the bacteriophage protein.
 52. Avector comprising an expression control sequence operably linked to anucleic acid sequence encoding a stabilized polypeptide, wherein saidstabilized polypeptide comprises a bioactive peptide and a firststabilizing group coupled to one of said bioactive peptide's termini,wherein said first stabilizing group is heterologous to the bioactivepeptide and lacks the capacity to participate in the formation of anintramolecular disulfide bond within the polypeptide.
 53. The vector ofclaim 52, wherein said stabilized polypeptide further comprises a secondstabilizing group coupled to the other terminus of said bioactivepeptide, wherein the second stabilizing group is heterologous to thebioactive peptide.
 54. The vector of claim 52, wherein said vectorfurther comprises a tightly regulable expression control sequenceoperably linked to said nucleic acid sequence encoding said stabilizedpolypeptide.
 55. The vector of claim 54, wherein said tightly regulableexpression control sequence is from a wild-type E. coli lacpromoter/operator region.
 56. The vector of claim 54, wherein saidexpression control sequence contains the auxiliary operator O3, the CAPbinding region, the −35 promoter site, the −10 promoter site, theoperator O1, lacZ Shine-Dalgarno sequence, and a spacer region.
 57. Thevector of claim 56, wherein said spacer region is 5 to 10 nucleotides inlength.
 58. The vector of claim 52, wherein said vector is pLAC11,represented by ATCC Accession No.
 207108. 59. A plurality of vectors,wherein each said vector comprises a nucleic acid sequence encoding apolypeptide, said polypeptide comprising a randomized peptide and afirst stabilizing group coupled to one of said randomized peptide'stermini, wherein said first stabilizing group lacks the capacity toparticipate in the formation of an intramolecular disulfide bond withinthe polypeptide, and wherein said plurality of vectors comprises atleast two different vectors, each of said at least two vectors encodinga different polypeptide.
 60. The plurality of vectors of claim 59,wherein said polypeptide further comprises a second stabilizing groupcoupled to the other terminus of said randomized peptide.
 61. Theplurality of vectors of claim 59, wherein each said vector furthercomprises a tightly regulable expression control sequence operablylinked to said nucleic acid sequence encoding said polypeptide.
 62. Theplurality of vectors of claim 59, wherein said plurality comprises atleast 50 different vectors, each of said at least 50 vectors encoding adifferent polypeptide.
 63. The plurality of vectors of claim 59, whereinsaid plurality comprises at least 1×10⁶ different vectors, each of saidat least 1×10⁶ vectors encoding a different polypeptide.
 64. A pluralityof host cells collectively containing the plurality of vectors of claim48.
 65. The host cells of claim 64, wherein said host cells areprokaryotic.
 66. The host cells of claim 64, wherein said host cells areeukaryotic.
 67. A method for selecting bioactive peptides, said methodcomprising: a) expressing a plurality of stabilized polypeptides,wherein each said stabilized polypeptide is encoded by a differentvector, and wherein each said stabilized polypeptide comprises arandomized peptide and a first stabilizing group coupled to one of saidrandomized peptide's termini, wherein said first stabilizing group lacksthe capacity to participate in the formation of an intramoleculardisulfide bond within the polypeptide, and wherein said plurality ofstabilized polypeptide comprises at least two different polypeptides;and b) screening each said stabilized polypeptide of said plurality fora bioactivity; and c) selecting said bioactive peptides with saidbioactivity.
 68. The method of claim 67, wherein said stabilizedpolypeptide further comprises a second stabilizing group coupled to theother terminus of said randomized peptide.
 69. The method of claim 67,wherein said plurality of stabilized polypeptides is expressed ineukaryotic cells.
 70. The method of claim 69, wherein said eukaryoticcells are cancer cells and said bioactivity is a complete or partialinhibition of cell division, induction of apoptosis, or cell toxicity.71. The method of claim 70, said method further comprising testing saidselected bioactive peptides for bioactivity in normal cells comparedwith said cancer cells.
 72. The method of claim 69, wherein saideukaryotic cells are stem cells or cord blood cells and said bioactivityis an increase in cell growth rate.
 73. A polypeptide comprising abioactive peptide and a stabilizing group coupled to one or both of saidbioactive peptide's termini, wherein said stabilizing group isheterologous to said bioactive peptide and consists of Xaa_(n)-Pro-Pro-or -Pro-Pro-Xaa_(n), wherein Xaa is any amino acid and n=1 or
 2. 74. Thepolypeptide of claim 73, wherein a heterologous stabilizing group iscoupled to both of said bioactive peptide's termini.
 75. The polypeptideof claim 73, wherein the Xaa residue of said heterologous stabilizinggroup is different on each end of said bioactive peptide.
 76. Thepolypeptide of claim 73, wherein said bioactive peptide is selected fromthe group consisting of insulin, glucagon, calcitonin, somatostatin,gonadotrophin, and secretin.
 77. A polypeptide comprising a bioactivepeptide and a first stabilizing group coupled to one terminus of saidbioactive peptide, wherein said bioactive peptide is 50 or fewer aminoacids in length, and wherein said first stabilizing group is human serumalbumin or a fragment thereof.
 78. The polypeptide of claim 77, whereinsaid polypeptide further comprises a second stabilizing group coupled tothe other terminus of said bioactive peptide.